Bioavailability 3-heteroarylidenyl-2-indolinones active as protein tyrosine kinase inhibitors

ABSTRACT

The present invention relates to novel 3-hetero-arylidenyl-2-indolinone compounds and physiologically acceptable salts and prodrugs thereof which have improved hydrosolubility and which are expected to modulate the activity of protein tyrosine kinases and therefore should be useful in the prevention and treatment of protein tyrosine kinase related cellular disorders such as cancer.

RELATED APPLICATIONS

[0001] This application is related to and claims priority fromprovisional application serial number 60/050,412 dated Jun. 20, 1997,and provisional application serial number 60/059,336, dated Sep. 19,1997, both of which are incorporated by reference as if fully set forthherein.

INTRODUCTION

[0002] The present invention relates generally to organic chemistry,biochemistry, pharmacology and medicine. More particularly, it relatesto novel heterocyclic compounds, and their physiologically acceptablesalts and prodrugs, which modulate the activity of protein tyrosinekinases (“PTKs”) and, therefore, are expected to exhibit a salutaryeffect against disorders related to abnormal PTK activity.

BACKGROUND OF THE INVENTION

[0003] The following is offered as background information only and isnot admitted to be prior art to the present invention.

[0004] Growth factor receptors are cell-surface proteins. When bound bya growth factor ligand, growth factor receptors are converted to anactive form which interacts with proteins on the inner surface of a cellmembrane. This leads to phosphorylation on tyrosine residues of thereceptor and other proteins and to the formation inside the cell ofcomplexes with a variety of cytoplasmic signaling molecules that, inturn, affect numerous cellular responses such as cell division(proliferation), cell differentiation, cell growth, expression ofmetabolic effects to the extracellular microenvironment, etc. For a morecomplete discussion, see Schlessinger and Ullrich, Neuron, 9:303-391(1992) which is incorporated by reference, including any drawings, as iffully set forth herein.

[0005] Growth factor receptors with PTK activity, known as receptortyrosine kinases (“RTKs”), comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.These RTKs consist of an extracellular glycosylated ligand bindingdomain, a transmembrane domain and an intracellular cytoplasmiccatalytic domain that can phosphorylate tyrosine residues on proteins.

[0006] Another RTK subfamily consists of insulin receptor (IR),insulin-like growth factor I receptor (IGF-1R) and the insulin receptorrelated receptor (IRR). IR and IGF-1R interact with insulin, IGF-I andIGF-II to form a heterotetramer of two entirely extracellularglycosylated α subunits and two β subunits which cross the cell membraneand which contain the tyrosine kinase domain.

[0007] A third RTK subfamily is referred to as the platelet derivedgrowth factor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ,CSFIR, c-kit and c-fms. These receptors consist of glycosylatedextracellular domains composed of variable numbers of immunoglobin-likeloops and an intracellular domain wherein the tyrosine kinase domains isinterrupted by unrelated amino acid sequences.

[0008] Another group which, because of its similarity to the PDGFRsubfamily, is sometimes subsumed in the later group is the fetus liverkinase (“flk”) receptor subfamily. This group is believed to be made ofup of kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1),flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

[0009] One further member of the tyrosine kinase growth factor receptorfamily is the group known as the fibroblast growth factor(“FGF”)receptors. This group consists of four receptors, FGFR1-4, andseven ligands, FGF1-7. While not yet well defined, it appears that thereceptors consist of a glycosylated extracellular domain containing avariable number of immunoglobin-like loops and an intracellular domainin which the PTK sequence is interrupted by regions of unrelated aminoacid sequences.

[0010] A more complete listing of the known RTK subfamilies is describedin Plowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

[0011] In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases”. This latter designation, abbreviated “CTK”, will beused in this disclosure. CTKs do not contain extracellular andtransmembrane domains. At present, over 24 CTKs in 11 subfamilies (Src,Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have beenidentified. The Src subfamily appear so far to be the largest group ofCTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For amore detailed discussion of CTKs, see Bolen, Oncogene, 8:2025-2031(1993), which is incorporated by reference, including any drawings, asif fully set forth herein.

[0012] Both RTKs and CTKs have been implicated in a host of pathogenicconditions including, significantly, cancer. Others include, withoutlimitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis,angiogenesis and a variety of renal disorders.

[0013] With regard to cancer, two of the major hypotheses advanced toexplain the excessive cellular proliferation that drives tumordevelopment relate to functions known to be PTK regulated. That is, ithas been suggested that malignant cell growth results from a breakdownin the mechanisms that control cell division and/or differentiation. Ithas been shown that the protein products of a number of proto-oncogenesare involved in the signal transduction pathways that regulate cellgrowth and differentiation. These protein products of proto-oncogenesinclude the extracellular growth factors, transmembrane growth factorPTK receptors (RTKs) and cytoplasmic PTKs (CTKs), discussed above.

[0014] In view of the apparent link between PTK-related cellularactivities and a number of human disorders, it is no surprise that agreat deal of effort is being expended in an attempt to identify ways tomodulate PTK activity. Some of these have involved biomimetic approachesusing large molecules patterned on those involved in the actual cellularprocesses; e.g., mutant ligands (U.S. Pat. No. 4,966,849); solublereceptors and antibodies (App. No. WO 94/10202, Kendall and Thomas,Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature,362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry,33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, etal., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys.,152:448-57)) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660;WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al.,Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).

[0015] More recently, attempts have been made to identify smallmolecules which act as PTK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinylene-azaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP App. No. 0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO91/15495) have all been described as PTK inhibitors with potentialutility for the treatment of cancer.

[0016] An area in need of improvement with regard to PTK-activecompounds is their bioavailability in vivo. It is not uncommon for amolecule to exhibit good PTK modulating activity in vitro, where it canbe placed in immediate proximity to the PTK of interest, but to havesubstantially less, sometimes no, activity in vivo. Without being boundto a particular theory, applicants believe that this phenomenon may bedue to the fact that many molecules of interest as modulators of PTKactivity, including indolinones, tend to be lipophilic. However, in manycases, the region where the PTKs reside and perform their function areaqueous in nature. Thus, the compounds might not be capable of reachingthe active site. Improving the hydrosolubility of indolinones could leadto compounds with improved bioavailiabilty and thereby improved PTKmodulation in vivo.

SUMMARY OF THE INVENTION

[0017] Our efforts to identify small organic molecules which exhibitimproved hydrosolubility while maintaining their ability to modulate PTKactivity and which, therefore, should be useful it the treatment andprevention of disorders driven by abnormal TK activity, has led us tothe discovery of a family of novel heterocyclic compounds which exhibitimproved hydrosolubility and still have the desired ability to modulatePTK activity and which are the subject of this invention. Thus, thepresent invention relates generally to novel3-heteroarylidenyl-2-indolinones which have improved hydrosolubility andwhich modulate the activity of both receptor (RTK) and non-receptor(CTK) protein tyrosine kinases (PTKs). In addition, the presentinvention relates to the preparation and use of pharmacologicalcompositions of the disclosed compounds and their physiologicallyacceptable salts and prodrugs in the treatment or prevention of PTKdriven disorders such as, by way of example and not limitation, cancer,diabetes, hepatic cirrhosis, atherosclerosis, angiogenesis and renaldisease.

[0018] A “3-heteroarylidenyl-2-indolinone” refers to a chemical compoundhaving a “heteroaryl” group, as defined below, bonded to a carbon-carbondouble bond, the other end of which double bond is bonded to the ringcarbon of the pyrrolidone ring of an indolin-2-one.

[0019] As used herein, “lipophilic” refers to molecules which have anaffinity for, or capability of dissolving in, lipids; i.e., non-watersoluble oils, fats, sterols, triglycerides and the like.

[0020] The term “hydrosoluble” and “hydrosolubility” refer to moleculeswhich have an affinity for, or capability of dissolving in, aqueoussolutions; i.e., solutions consisting primarily of water.

[0021] The terms “indolin-2-one”, “2-indolinone” and “2-oxindole allrefer to a six-member aryl fused through two adjacent ring carbons to apyrrolidone ring at the carbon adjacent to the ring nitrogen and to thecarbon next to that carbon.

[0022] When D, E, F or G is nitrogen the bicyclic ring is properlytermed as “azaindolin-2-one.” However, for the purposes of thisdisclosure the term indolin-2-one, 2-indolinone or 2-oxindole will beunderstood to incorporate the nitrogen-containing compounds as well.

[0023] A “pyrrolidone” ring has the structure:

[0024] As used herein, the term “heteroarylidenyl” group refers to thegroup consisting of the double bond and the heteroaryl group and R²bonded to it.

[0025] A “pharmacological composition” refers to a mixture of one ormore of the compounds described herein, or physiologically acceptablesalts or prodrugs thereof, with other chemical components, such asphysiologically acceptable carriers and excipients. The purpose of apharmacological composition is to facilitate administration of acompound to an organism.

[0026] A “prodrug” refers to an agent which is converted into the parentdrug in vivo. Prodrugs are often useful because, in some situations,they may be easier to administer than the parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmacologicalcompositions over the parent drug. An example, without limitation, of aprodrug would be a compound of the present invention wherein it isadministered as an ester (the “prodrug”) to facilitate transmittalacross a cell membrane where water solubility is not beneficial, butthen it is metabolically hydrolyzed to the carboxylic acid once insidethe cell where water solubility is beneficial.

[0027] As used herein, an “ester” is a carboxy group, as defined herein,wherein “R” is any of the listed groups other than hydrogen.

[0028] As used herein, a “physiologically acceptable carrier” refers toa carrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

[0029] An “excipient” refers to an inert substance added to apharmacological composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0030] 1. The Compounds

[0031] A. General Structural Features.

[0032] In one aspect, this invention relates to a3-hetero-arylidenyl-2-indolinone compound which is substituted with oneor more groups which have an affinity for combining with water and whichtherefore improve the hydrosolubility of the3-heteroarylidenyl-2-indolinone.

[0033] By “affinity for combining with water” is meant that the group,in the presence of water, interacts electronically with water moleculesthrough, but not limited to, such mechanisms as ionization, hydrogenbonding and hydration to form complexes with the water molecules whichcan improve the solubility of the entire molecule in water.

[0034] By “improve the hydrosolubility” is meant that the compoundsubstituted with one or more of the indicated groups is more soluble inwater than the same molecule without the indicated groups.

[0035] In another aspect, the present invention relates to3-heteroarylidenyl-2-indolinones having the chemical structure shown inFormula 1:

[0036] A is selected from the group consisting of nitrogen, oxygen andsulfur and it is understood that when A is oxygen or sulfur, R³ does notexist.

[0037] B, D, E, F and G are independently selected from the groupconsisting of carbon and nitrogen and it is understood that when B, D,E, F or G is nitrogen, R⁴, R⁵, R⁶ or R⁷, respectively, do not exist.

[0038] Z is selected from the group consisting of oxygen, sulfur andNR¹¹.

[0039] R¹¹ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, carbonyl, C-carboxy,O-carboxy, C-amido, guanyl, sulfonyl and trihalomethanesulfonyl.

[0040] R¹ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, alkenyl, alkynyl, aryl, hydroxy, alkoxy, C-carboxy, C-amido,trihalomethanecarbonyl, trihalomethanesulfonyl and sulfonyl.

[0041] R² is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl and halogen.

[0042] When A is nitrogen, R³ is selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, carbonyl,C-carboxy, O-carboxy, C-amido, guanyl, sulfonyl andtrihalomethanesulfonyl.

[0043] One or two of R⁴, R⁵, R⁶ and R⁷ are independently selected fromthe group consisting of —NR⁸R⁹, —J(CH₂)_(m)—NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q,—N═CNR⁸R⁹ and —NHR¹⁰.

[0044] J is selected from the group consisting of oxygen, nitrogen andsulfur.

[0045] The subscript m can be 0, 1, 2 or 3.

[0046] Y is selected from the group consisting of —NH and oxygen.

[0047] Q is selected from the group consisting of hydroxy, alkoxy,aryloxy, amino, N-hydroxylamino, O-carboxy, NR⁸R⁹ and N-peptidyl. R⁸ andR⁹ are independently selected from the group consisting of hydrogen,alkyl, C-carboxy, C-peptidyl and, combined, a five-member or 6-memberheteroalicyclic group containing at least one nitrogen.

[0048] R¹⁰ is a polyhydroxyalkyl group.

[0049] The remaining groups are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido,N-Sulfonamido, trihalomethanesulfonyl, carbonyl, C-carboxy, O-carboxy,C-amido, N-amido, cyano, nitro, halo, O-thiocarbamyl, N-thiocarbamyl,guanyl and phosphonyl; and,

[0050] R⁴ and R⁵ may combine to form a six-member cycloalkyl, heteroarylor heteroalicyclic ring.

[0051] As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms (whenever anumerical range; e.g. “1-20”, is stated herein, it means that the group,in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms,3 carbon atoms, etc. up to and including 20 carbon atoms). Morepreferably, it is a medium size alkyl having 1 to 10 carbon atoms. Mostpreferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkylgroup may be substituted or unsubstituted. When substituted, thesubstituent group(s) is preferably one or more individually selectedfrom cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfonamido,trihalomethane-sulfonamido, silyl, guanyl, guanidino, ureido, amino andNR¹²R¹³, wherein R¹² and R¹³ are independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, sulfonyl,trihalomethysulfonyl and, combined, a five- or six-memberheteroalicyclic ring.

[0052] A “cycloalkyl” group refers to an all-carbon monocyclic or fusedring (i.e., rings which share an adjacent pair of carbon atoms) groupwherein one of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more individually selectedfrom alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, nitro, guanyl, ureido, guanidino,amino and NR¹²R¹³, with R¹² and R¹³ being as defined herein.

[0053] An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond.

[0054] An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond.

[0055] An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more selectedfrom halo, trihalomethyl, alkyl, alkenyl, alkynyl, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, amino andNR¹²R¹³ wherein R¹² and R¹³ are previously defined herein.

[0056] As used herein, a “heteroaryl” group refers to a monocyclic orfused ring (i.e., rings which share an adjacent pair of atoms) grouphaving in the ring(s) one or more atoms selected from the groupconsisting of nitrogen, oxygen and sulfur and, in addition, having acompletely conjugated pi-electron system. Examples, without limitation,of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline,purine and carbazole. The heteroaryl group may be substituted orunsubstituted. When substituted, the substituted group(s) is preferablyone or more selected from alkyl, cycloalkyl, halo, trihalomethyl,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano,nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxy, O-carboxy,sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,trihalomethanesulfonamido, amino and NR¹²R¹³ where R¹² and R¹³ arepreviously defined herein.

[0057] A “heteroalicyclic” group refers to a monocyclic or fused ringgroup having in the ring(s) one or more atoms selected from the groupconsisting of nitrogen, oxygen and sulfur. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. The heteroalicyclic ring may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably one or more selected from alkyl, cycloaklyl, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, sulfinyl,sulfonyl, S-sulfonamido, N-sulfonamido, C-amido, N-amido, ureido,guanyl, guanidino, amino and N¹²R¹³ where R¹² and R¹³ are previouslydefined herein.

[0058] A “hydroxy” group refers to an —OH group.

[0059] An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkylgroup, as defined herein.

[0060] An “aryloxy” group refers to both an -O-aryl and an —O-heteroarylgroup, as defined herein.

[0061] A “thiohydroxy” group refers to an -SH group.

[0062] A “thioalkoxy” group refers to both an S-alkyl and an—S-cycloalkyl group, as defined herein.

[0063] A “thioaryloxy” group refers to both an -S-aryl and an—S0-heteroaryl group, as defined herein.

[0064] A “carbonyl” group refers to a —C(═O)—R″ group, where R″ isselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heteroalicyclic (bondedthrough a ring carbon), as defined herein.

[0065] An “aldehyde” group refers to a carbonyl group where R″ ishydrogen.

[0066] A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ asdefined herein.

[0067] A “trihalomethanecarbonyl” group refers to a X₃CC (═O)— groupwith X as defined herein.

[0068] A “C-carboxy” group refers to a —C(═O)O—R″ groups, with R″ asdefined herein.”An “O-carboxy” group refers to a R″C(═O)O— group, withR″ as defined herein.

[0069] A “carboxylic acid” group refers to a C-carboxy group in which R″is hydrogen.

[0070] A “halo” group refers to fluorine, chlorine, bromine or iodine.

[0071] A “trihalomethyl” group refers to a —CX₃ group wherein X is ahalo group as defined herein.

[0072] A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groupswith X as defined above.

[0073] A “trihalomethanesulfonamido” group refers to a X₃CS (═O)₂NR¹²—group with X and R¹² as defined herein.

[0074] A “sulfinyl” group refers to a —S(═O)—R″ group, with R″ asdefined herein.

[0075] A “sulfonyl” group refers to a —S(═O)₂R″ group, with R″ asdefined herein.

[0076] An “S-sulfonamido” group refers to a —S(═O)₂NR¹²R¹³, with R¹² andR¹³ as defined herein.

[0077] An “N-Sulfonamido” group refers to a R¹²S(═O)₂NR¹³— group, withR¹² and R¹³ as defined herein.

[0078] An “O-carbamyl” group refers to a —OC(═O)NR¹²R¹³ group with R¹²and R¹³ as defined herein.

[0079] An “N-carbamyl” group refers to a R¹²OC(═O)NR¹³— group, with R¹²and R¹³ as defined herein.

[0080] An “O-thiocarbamyl” group refers to a —OC(═S)NR¹²R¹³ group withR¹² and R¹³ as defined herein.

[0081] An “N-thiocarbamyl” group refers to a R¹²OC(═S)NR¹³— group, withR¹² and R¹³ as defined herein.

[0082] An “amino” group refers to an —NR¹²R¹³ group, with R¹² and R¹³ asdefined herein.

[0083] A “C-amido” group refers to a —C(═O)NR¹²R¹³ group with R¹² andR¹³ as defined herein.

[0084] An “N-amido” group refers to a R¹²C(═O)NR¹³— group, with R¹² andR¹³ as defined herein.

[0085] A “quaternary ammonium” group refers to a —⁺NHR¹²R¹³ groupwherein R¹² and R¹¹ are independently selected from the group consistingof alkyl, cycloalkyl, aryl, and heteroaryl.

[0086] A “ureido” group refers to a —NR¹²C(═O)NR¹³R¹⁴ group, with R¹²and R¹³ as defined herein and R¹⁴ defined the same as R¹² and R¹³.

[0087] A “guanidino” group refers to a —R¹²NC(═N)NR¹³R¹⁴ group, withR¹², R¹³ and R¹⁴ as defined herein.

[0088] A “guanyl” group refers to a R¹²R¹³NC(═N)— group, with R¹² andR¹³ as defined herein.

[0089] A “nitro” group refers to a —NO₂ group.

[0090] A “cyano” group refers to a —C≡N group.

[0091] A “silyl” group refers to a —Si(R″)₃ group, with R″ as definedherein.

[0092] An “N-hydroxylamino” group refers to a —NHOR″ group, with R″ asdefined herein.

[0093] A “polyhydroxyalkyl” group refers to a 1 to 8 carbon, preferablya 1 to 4 carbon straight chain alkyl group substituted with 2 or more,preferrably 3 or more, hydroxyl groups.

[0094] A “peptidyl” group generally refers to a group formed by theinteraction between the amino groups and the carboxy groups of aminoacids. A peptidyl group has the general formula:

[0095] wherein the Rs may be the same or different. The amino acid onthe left hand side of the above formula is referred to as the N-terminalamino acid residue and the amino acid on the right hand side is referredto as the C-terminal amino acid residue.

[0096] An “N-peptidyl” group refers to a peptidyl group which is bondedthrough the N-terminal amino acid to a non-amino acid molecule.

[0097] A “C-peptidyl” group refers to a peptidyl group which is bondedthrough the C-terminal amino acid to a non-amino acid molecule.

[0098] B. Preferred Structural Features.

[0099] Preferred structural features of this invention are those inwhich:

[0100] R¹ is selected from the group consisting of hydrogen, alkyl,hydroxy, alkoxy and C-carboxy;

[0101] Z is selected from the group consisting of sulfur and oxygen;

[0102] R² is selected from the group consisting of hydrogen and alkyl;

[0103] A and B are nitrogen;

[0104] R³ is selected from the group consisting of hydrogen and alkyl;and,

[0105] R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, alkyl, aryl, heteroaryl, C-carboxy, alkoxy, cyano, andC-carboxy.

[0106] Additional preferred structures of the present invention arethose in which:

[0107] A is sulfur; and,

[0108] R⁴ and R⁵, combined, form a six-member cycloalkyl, heteroaryl orheteroalicyclic ring.

[0109] Still further preferred embodiments of the present invention arethose in which:

[0110] A is nitrogen;

[0111] B is carbon;

[0112] R¹ is hydrogen; and,

[0113] R⁴ and R⁵ are lower alkyl.

[0114] The chemical formulae referred to herein may exhibit thephenomena of tautomerism and structural isomerism. For example, thecompounds described herein may adopt a cis or trans configuration aboutthe double bond connecting the indolin-2-one moiety to the heteroarylmoiety or they may be a mixture of cis and trans. This inventionencompasses any tautomeric or structural isomeric form and mixturesthereof which possess the ability to modulate RTK and/or CTK activityand is not limited to any one tautomeric or structural isomeric form.

[0115] As used herein, the term “cis” refers to the structuralconfiguration wherein the heteroaryl group is on the same side of thedouble bond connecting it to the indolin-2-one ring as the 2-oxygengroup of the indolin-2-one.

[0116] As used herein, the term “trans” refers to the structuralconfiguration wherein the heteroaryl group is on the opposite side ofthe double bond connecting it to the indolin-2 -one ring as the 2-oxygengroup of the indolin-2-one.

[0117] 2. The Biochemistry

[0118] In yet another embodiment, this invention relates to a method forthe modulation of the catalytic activity of PTKs comprisingadministering a compound of this invention or a physiologicallyacceptable salt or a prodrug thereof to a PTK.

[0119] By “PTK” is meant both RTKs and CTKs; i.e., the modulation ofboth RTK signal transduction and CTK signal transduction is contemplatedby this invention.

[0120] The term “method” refers to manners, means, techniques andprocedures for accomplishing a given task including, but not limited to,those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby, practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

[0121] As used herein, the term “modulation” or “modulating” refers tothe alteration of the catalytic activity of RTKs and/or CTKs. Inparticular, modulating refers to the activation of the catalyticactivity of RTKs and/or CTKs, more preferably the activation orinhibition of the catalytic activity of RTKs and/or CTKs, depending onthe concentration of the compound administered or, more preferablystill, the inhibition of the catalytic activity of RTKs and/or CTKs.

[0122] The term “catalytic activity” as used herein refers to the rateof phosphorylation of tyrosine under the influence, direct or indirectof RTKs and/or CTKS.

[0123] The term “administering” as used herein refers to a method forbringing a compound of this invention and a target PK together in such amanner that the compound can affect the catalytic activity of the PKeither directly; i.e., by interacting with the kinase itself, orindirectly; i.e. by interacting with another molecule on which thecatalytic activity of the kinase is dependent. As used herein,administration can be accomplished either in vitro, i.e. in a test tube,or in vivo, i.e. in cells or tissues of a living organism. Thus, the TKmediated disorders which are the object of this invention can bestudied, prevented or treated by the methods set forth herein whetherthe cells or tissues of the organism exist within the organism oroutside the organism. Cells existing outside the organism can bemaintained or grown in cell culture dishes. In this context, the abilityof a particular compound to affect a PTK related disorder can bedetermined; i.e., the IC50 of the compound, defined below, before theuse of the compounds in more complex living organisms is attempted. Forcells outside the organism, multiple methods exist, and are well-knownto those skilled in the art, to administer compounds including, but notlimited to, direct cell microinjection and numerous transmembranecarrier techniques. For cells harbored within a living organism, myriadmethods also exist, and are likewise well-known to those skilled in theart, to administer compounds including, but not limited to, oral,parenteral, dermal and aerosol applications.

[0124] RTK mediated signal transduction is initiated by extracellularinteraction with a specific growth factor (ligand), followed by receptordimerization, transient stimulation of the intrinsic protein tyrosinekinase activity and phosphorylation. Binding sites are thereby createdfor intracellular signal transduction molecules and lead to theformation of complexes with a spectrum of cytoplasmic signalingmolecules that facilitate the appropriate cellular response (e.g., celldivision, metabolic effects to the extracellular microenvironment). See,Schlessinger and Ullrich, 1992, Neuron 9:303-391.

[0125] It has been shown that tyrosine phosphorylation sites in growthfactor receptors function as high-affinity binding sites for SH2 (srchomology) domains of signaling molecules. Fantl et al., 1992, Cell69:413-423; Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785);Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science252:668-678. Several intracellular substrate proteins that associatewith RTKs have been identified. They may be divided into two principalgroups: (1) substrates which have a catalytic domain; and (2) substrateswhich lack such domain but serve as adapters and associate withcatalytically active molecules. Songyang et al., 1993, Cell 72:767-778.The specificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

[0126] PTK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis,arthritis and diabetic retinopathy (or other disorders related touncontrolled angiogenesis and/or vasculogenesis).

[0127] A precise understanding of the mechanism by which the compoundsof this invention inhibit PTKs is not required in order to practice thepresent invention. However, while not being bound to any particularmechanism or theory, it is believed that the compounds interact with theamino acids of the catalytic region of PTKs. PTKs typically possess abi-lobate structure wherein ATP appears to bind in the cleft between thetwo lobes in a region where the amino acids are conserved among PTKs.Inhibitors of PTKs are believed to bind by non-covalent interactionssuch as hydrogen bonding, van der Waals forces and ionic interactions inthe same general region where the aforesaid ATP binds to the PTKs. Morespecifically, it is thought that the indolinone component of thecompounds of this invention binds in the general space normally occupiedby the adenine ring of ATP. Specificity of a particular molecule for aparticular PTK could arise as the result of additional interactionsbetween the various substituents on the indolinone core with amino aciddomains specific to particular PTKs. Thus, different indolinonesubstituents may contribute to preferential binding to particular PTKs.The ability to select those compounds active at different ATP (or othernucleotide) binding sites makes the compounds useful for targeting anyprotein with such a site; i.e., not only PTKs but serine/threoninekinases and protein phosphatases as well. Thus, the compounds disclosedherein have utility for in vitro assays on such proteins and for in vivotherapeutic effects through such proteins.

[0128] Thus, in another aspect, this invention relates to a method fortreating or preventing a PTK related disorder by administering atherapeutically effective amount of a compound of this invention or asalt or a prodrug thereof to an organism.

[0129] As used herein, “PTK related disorder,” “PTK driven disorder,”and “abnormal PTK activity” all refer to a disorder characterized byinappropriate or over-activity of PTKs, which can be either RTKs orCTKs. Inappropriate activity refers to either: (1) PTK expression incells which normally do not express PTKs; (2) increased PTK expressionleading to unwanted cell proliferation, differentiation and/or growth;or, (3) decreased PTK expression leading to unwanted reductions in cellproliferation, differentiation and/or growth. Overactivity of PTKsrefers to either amplification of the gene encoding a particular PTK orproduction of a level of PTK activity which can correlate with a cellproliferation, differentiation and/or growth disorder (that is, as thelevel of the PTK increases, the severity of one or more of the symptomsof the cellular disorder increases).

[0130] As used herein, the terms “prevent”, “preventing” and“prevention” refer to a method for barring an organism from the firstplace acquiring an PTK mediated cellular disorder.

[0131] As used herein, the terms “treat”, “treating” and “treatment”refer to a method of alleviating or abrogating the PTK mediated cellulardisorder and/or its attendant symptoms. With regard particularly tocancer, these terms simply mean that the life expectancy or anindividual affected with a cancer will be increased or that one or moreof the symptoms of the disease will be reduced.

[0132] As used herein, the term “cancer” refers to various types ofmalignant neoplasms, most of which can invade surrounding tissues, andmay metastasize to different sites, as defined by Stedman's MedicalDictionary 25th edition (Hensyl ed. 1990). Examples of cancers which maybe treated by the present invention include, but are not limited to,brain, ovarian, colon, prostate, kidney, bladder, breast, lung, oral andskin cancers which exhibit inappropriate PTK activity. These types ofcancers can be further characterized. For example, brain cancers includeglioblastoma multiforme, anaplastic astrocytoma, astrocytoma,ependymoma, oligodendroglioma, medulloblastoma, meningioma, sarcoma,hemangioblastoma, and pineal parenchymal. Skin cancers include melanomaand Kaposi's sarcoma.

[0133] The term “organism” refers to any living entity comprised of atleast one cell. A living organism can be as simple as, for example, asingle eukariotic cell or as complex as a mammal, including a humanbeing.

[0134] The term “therapeutically effective amount” as used herein refersto that amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor; (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis; (3) inhibiting to some extent(that is slowing to some extent, preferably stopping) tumor growth;and/or, (4) relieving to some extent (or preferably eliminating) one ormore symptoms associated with the cancer.

[0135] This invention is therefore directed to compounds which modulatePTK signal transduction by affecting the enzymatic activity of the RTKsand/or CTKs and thereby interfering with the signal transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate the RTK and/or CTK mediated signal transductionpathways as a therapeutic approach to cure many kinds of solid tumors,including but not limited to carcinoma, sarcoma, erythroblastoma,glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma.Indications may include, but are not limited to brain cancers, bladdercancers, ovarian cancers, gastric cancers, pancreas cancers, coloncancers, blood cancers, lung cancers, bone cancers and leukemias.

[0136] Further examples, without limitation, of the types of disordersrelated to unregulated PTK activity that the compounds described hereinmay be useful in preventing, treating and studying, are cellproliferative disorders, fibrotic disorders and metabolic disorders.

[0137] Cell proliferative disorders which may be prevented, treated orfurther studied by the present invention include cancers, blood vesselproliferative disorders and mesangial cell proliferative disorders.

[0138] Blood vessel proliferative disorders refer to angiogenic andvasculogenic disorders generally resulting in abnormal proliferation ofblood vessels. The formation and spreading of blood vessels, orvasculogenesis and angiogenesis, respectively, play important roles in avariety of physiological processes such as embryonic development, corpusluteum formation, wound healing and organ regeneration. They also play apivotal role in cancer development. Other examples of blood vesselproliferation disorders include arthritis, where new capillary bloodvessels invade the joint and destroy cartilage, and ocular diseases,like diabetic retinopathy, where new capillaries in the retina invadethe vitreous, bleed and cause blindness. Conversely, disorders relatedto the shrinkage, contraction or closing of blood vessels, such asrestenosis, are also implicated.

[0139] Fibrotic disorders refer to the abnormal formation ofextracellular matrices. Examples of fibrotic disorders include hepaticcirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosisis characterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. Hepatic cirrhosis cancause diseases such as cirrhosis of the liver. An increasedextracellular matrix resulting in a hepatic scar can also be caused byviral infection such as hepatitis. Lipocytes appear to play a major rolein hepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

[0140] Mesangial cell proliferative disorders refer to disorders broughtabout by abnormal proliferation of mesangial cells. Mesangialproliferative disorders include various human renal diseases, such asglomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes, transplant rejection, andglomerulopathies. For instance, PDGFR has been implicated in themaintenance of mesangial cell proliferation. Floege et al., 1993, KidneyInternational 43:47S-54S.

[0141] As noted previously, PTKs have been associated with cellproliferative disorders. For example, some members of the RTK familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torpet al., 1992, APMIS 100:713-719); HER2/neu (Slamon et al., 1989, Science244:707-712) and PDGFR (Kumabe et al., 1992, Oncogene, 7:627-633) areover-expressed in many tumors and/or are persistently activated byautocrine loops. In fact, in the most common and severe cancers thesereceptor over-expressions and autocrine loops have been demonstrated(Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci., 111:119-133;Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al.,1992, J. Clin. Invest. 90:1352-1360); (Lee and Donoghue, 1992, J. Cell.Biol., 118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak etal., supra). For example, the EGFR receptor has been associated withsquamous cell carcinoma, astrocytoma, glioblastoma, head and neckcancer, lung cancer and bladder cancer. HER2 has been associated withbreast, ovarian, gastric, lung, pancreas and bladder cancer. PDGFR hasbeen associated with glioblastoma, lung, ovarian, melanoma and prostate.The RTK c-met has been generally associated with hepatocarcinogenesisand thus hepatocellular carcinoma. Additionally, c-met has been linkedto malignant tumor formation. More specifically, the RTK c-met has beenassociated with, among other cancers, colorectal, thyroid, pancreaticand gastric carcinoma, leukemia and lymphoma. Additionally,over-expression of the c-met gene has been detected in patients withHodgkins disease, Burkitts disease, and the lymphoma cell line.

[0142] IGF-IR, in addition to being implicated in nutritional supportand in type-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while being integrally involved in the normal growthand differentiation of the nervous system, appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of the IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cellsof the bone marrow) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series ofrecent publications, Baserga even suggests that IGF-IR plays a centralrole in the mechanisms of transformation and, as such, could be apreferred target for therapeutic interventions for a broad spectrum ofhuman malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga,1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol.,14:4588-4595.

[0143] The association between abnormal RTK activity and disease are notrestricted to cancer, however. For example, RTKs have been associatedwith metabolic diseases like psoriasis, diabetes mellitus, woundhealing, inflammation, and neurodegenerative diseases. For example, EGFRhas been indicated in corneal and dermal wound healing. Defects in theInsulin-R and IGF-1R are indicated in type-II diabetes mellitus. A morecomplete correlation between specific RTKs and their therapeuticindications is set forth in Plowman et al., 1994, DN&P 7:334-339.

[0144] As noted previously, not only RTKs but CTKs as well including,but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr andyrk (reviewed by Bolen et al., 1992, FASEB J., 6:3403-3409) are involvedin the proliferative and metabolic signal transduction pathway and thuswere expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been demonstrated as an oncoprotein(pp60^(v-src)) in chicken. Moreover, its cellular homolog, theproto-oncogene pp60^(c-src) transmits oncogenic signals of manyreceptors. For example, over-expression of EGFR or HER2/neu in tumorsleads to the constitutive activation of pp60^(c-src), which ischaracteristic for the malignant cell but absent from the normal cell.On the other hand, mice deficient in the expression of c-src exhibit anosteopetrotic phenotype, indicating a key participation of c-src inosteoclast function and a possible involvement in related disorders.Similarly, Zap70 is implicated in T-cell signaling.

[0145] Finally, both RTKs and CTKs are currently suspected as beinginvolved in hyperimmune disorders.

[0146] 3. Pharmacologcal Compositions and Therapeutic Applications

[0147] A compound of the present invention, or its physiologicallyacceptable salt or prodrug, can be administered to a human patient perse, or in pharmacological compositions where it is mixed with suitablecarriers or excipient(s). Techniques for formulation and administrationof drugs may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition.

[0148] A. Routes Of Administration.

[0149] Suitable routes of administration may include, withoutlimitation, oral, rectal, transmucosal or intestinal administration orintramuscular, subcutaneous, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal or intranasal injections.

[0150] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a solid tumor, often in a depot or sustained releaseformulation.

[0151] Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with tumor-specificantibody. The liposomes will be targeted to and taken up selectively bythe tumor.

[0152] B. Composition/Formulation.

[0153] Pharmacological compositions of the compounds and thephysiologically acceptable salts and prodrugs thereof are preferredembodiments of this invention. Pharmacological compositions of thepresent invention may be manufactured by processes well known in theart; e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

[0154] Pharmacological compositions for use in accordance with thepresent invention thus may be formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

[0155] For injection, the compounds of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0156] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmacological preparations fororal use can be made with the use of a solid excipient, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0157] Dragée cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0158] Pharmacological compositions which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0159] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0160] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0161] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0162] Pharmacological compositions for parenteral administrationinclude aqueous solutions of the active compounds in water soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

[0163] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0164] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0165] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0166] The pharmacological compositions herein also may comprisesuitable solid or gel phase carriers or excipients. Examples of suchcarriers or excipients include but are not limited to calcium carbonate,calcium phosphate, various sugars, starches, cellulose derivatives,gelatin, and polymers such as polyethylene glycols.

[0167] Many of the PTK modulating compounds of the invention may beprovided as physiologically acceptable salts wherein the claimedcompound may form the negatively or the positively charged species.Examples of salts in which the compound forms the positively chargedmoiety include, without limitation, quaternary ammonium (definedelsewhere herein), salts such as the hydrochloride, sulfate, carbonate,lactate, tartrate, maleate, succinate, etc. formed by the reaction of anamino group with the appropriate acid. Salts in which the compound formsthe negatively charged species include, without limitation, the sodium,potassium, calcium and magnesium salts formed by the reaction of acarboxylic acid group in the molecule with the appropriate base (e.g.sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide(Ca (OH₂), etc.).

[0168] C. Dosage.

[0169] Pharmacological compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve its intended purpose.

[0170] More specifically, a therapeutically effective amount means anamount of compound effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

[0171] Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

[0172] For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. For example, a dose can be formulated in animalmodels to achieve a circulating concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of the PTK activity).Such information can be used to more accurately determine useful dosesin humans.

[0173] Toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1).

[0174] Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the kinase modulating effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data; e.g., the concentration necessary toachieve 50-90% inhibition of the kinase using the assays describedherein. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

[0175] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen which maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%.

[0176] In cases of local administration or selective uptake, theeffective local concentration of the drug may not be related to plasmaconcentration.

[0177] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

[0178] D. Packaging.

[0179] The compositions may, if desired, be presented in a pack ordispenser device, such as an FDA approved kit, which may contain one ormore unit dosage forms containing the active ingredient. The pack mayfor example comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions foradministration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of the labeling approved by the U.S. Foodand Drug Administration for prescription drugs or of an approved productinsert. Compositions comprising a compound of the invention formulatedin a compatible pharmaceutical carrier may also be prepared, placed inan appropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of a tumor, inhibition of angiogenesis, treatment of fibrosis,diabetes, and the like.

[0180] 4. Synthesis

[0181] The compounds of this invention, as well as the precursorindolin-2-ones and aldehydes, may be readily synthesized usingtechniques well known in the chemical arts. It will be appreciated bythose skilled in the art that other synthetic pathways for forming thecompounds of the invention are available and that the following isoffered by way of example and not limitation.

[0182] A. General Synthetic Procedure.

[0183] The following general methodology may be employed to prepare thecompounds of this invention:

[0184] The appropriately substituted indolin-2-one (1 equiv.), theappropriately substituted aldehyde (1.2 equiv.) and piperidine (0.1equiv.) are mixed with ethanol (1-2 ml/mmol 2-indolinone) and themixture is then heated at 90° C. for 3 to 5 hours After cooling, theprecipitate is filtered, washed with cold ethanol and dried to yield thetarget compound.

[0185] B. 2-oxindoles

[0186] The following examples show representative syntheses of severalof the 2-oxindole precursors to the compounds of this invention. These2-oxindoles, as well as the others claimed, will form the claimedcompounds by reaction with an appropriately substituted aldehyde underthe conditions described above. It is to be understood that thefollowing syntheses are provided by way of example only and are not tobe construed as limiting as to synthetic procedure or as to thecompounds described.

5-Amino-2-oxindole

[0187] 5-Nitro-2-oxindole (6.3 g) was hydrogenated in methanol over 10%palladium on carbon to give 3.0 g (60% yield) of the title compound as awhite solid.

5-Bromo-2-oxindole

[0188] 2-Oxindole (1.3 g) in 20 mL acetonitrile was cooled to −10° C.and 2.0 g N-bromosuccinimide was slowly added with stirring. Thereaction was stirred for 1 hour at −10° C. and 2 hours at 0° C. Theprecipitate was collected, washed with water and dried to give 1.9 g(90% yield) of the title compound.

4 -Methy-2-oxindole

[0189] Diethyl oxalate (30 mL) in 20 mL of dry ether was added withstirring to 19 g of potassium ethoxide suspended in 50 mL of dry ether.The mixture was cooled in an ice bath and 20 mL of 3-nitro-o-xylene in20 mL of dry ether was slowly added. The thick dark red mixture washeated to reflux for 0.5 hr, concentrated to a dark red solid, andtreated with 10% sodium hydroxide until almost all of the soliddissolved. The dark red mixture was treated with 30% hydrogen peroxideuntil the red color changed to yellow. The mixture was treatedalternately with 10% sodium hydroxide and 30% hydrogen peroxide untilthe dark red color was no longer present. The solid was filtered off andthe filtrate acidified with 6N hydrochloric acid. The resultingprecipitate was collected by vacuum filtration, washed with water, anddried under vacuum to give 9.8 g (45% yield) of2-methyl-6-nitrophenylacetic acid as an off-white solid. The solid washydrogenated in methanol over 10% palladium on carbon to give 9.04 g ofthe title compound as a white solid.

7-Bromo-5-chloro-2-oxindole

[0190] 5-Chloro-2-oxindole (16.8 g) and 19.6 g of N-bromosuccinimidewere suspended in 140 mL of acetonitrile and refluxed for 3 hours. Thinlayer chromatography (silica, ethyl acetate) at 2 hours of reflux showed5-chloro-2-oxindole or N-bromosuccinimide (Rf 0.8), product (Rf 0.85)and a second product (Rf 0.9) whose proportions did not change afteranother hour of reflux. The mixture was cooled to 10° C., theprecipitate was collected by vacuum filtration, washed with 25 mL ofethanol and sucked dry for 20 minutes in the funnel to give 14.1 g ofwet product (56% yield). The solid was suspended in 200 mL of denaturedethanol and slurry-washed by stirring and refluxing for 10 minutes. Themixture was cooled in an ice bath to 10° C. The solid product wascollected by vacuum filtration, washed with 25 mL of ethanol and driedunder vacuum at 40° C. to give 12.7 g (51% yield) of7-bromo-5-chloro-2-oxindole.

5-Fluoro-2-oxindole

[0191] 5-Fluoroisatin (8.2 g) was dissolved in 50 mL of hydrazinehydrate and refluxed for 1.0 hr. The reaction mixtures were then pouredin ice water. The precipitate was then filtered, washed with water anddried under vacuum oven afford the title compound.

5 -Nitro-2 -oxindole

[0192] 2-Oxindole (6.5 g) was dissolved in 25 mL concentrated sulfuricacid and the mixture maintained at −10 to −15° C. while 2.1 mL of fumingnitric acid was added dropwise. After the addition of the nitric acidthe reaction mixture was stirred at 0° C. for 0.5 hr and poured intoice-water. The precipitate was collected by filtration, washed withwater and crystallized from 50% acetic acid. The crystalline product wasthen filtered, washed with water and dried under vacuum to give 6.3 g(70%) of 5-nitro-2-oxindole.

5-Iodo-2-oxindole

[0193] 2-Oxindole (82.9 g) was suspended in 630 mL of acetic acid withmechanical stirring and the mixture cooled to 10° C. in an ice waterbath. Solid N-iodosuccinimide (175 g) was added in portions over 10minutes. After the addition was complete the mixture was stirred for 1.0hour at 10° C. The suspended solid which had always present became verythick at this time. The solid was collected by vacuum filtration, washedwith 100 mL of 50% acetic acid in water and then with 200 mL of waterand sucked dry for 20 minutes in the funnel. The product was dried undervacuum to give 93.5 g (36%) of 5-iodo-2-oxindole containing about 5%2-oxindole by proton NMR.

5-Methyl-2-oxindole

[0194] 5-Methylisatin (15.0 g) and 60 mL of hydrazine hydrate wereheated at 140 to 160° C. for 4 hours. Thin layer chromatography (ethylacetate:hexane 1:2, silica gel) showed no starting material remaining.The reaction mixture was cooled to room temperature, poured into 300 mLof ice water and acidified to pH 2 with 6N hydrochloric acid. Afterstanding at room temperature for 2 days the precipitate was collected byvacuum filtration, washed with water and dried under vacuum to give 6.5g (47% yield) of 5-methyl-2-oxindole.

5-Bromo-4-methyloxindole and 5,7-Dibromo-4-methyloxindole

[0195] 4-Methyl-2-oxindole (5 g) in 40 mL of acetonitrile was treatedwith 7.26 g of N-bromosuccinimide and stirred at room temperature for 4hours. Thin layer chromatography (ethyl acetate:hexane 1:2, silica gel)showed a mixture of 5-bromo (Rf 0.3) and 5,7-dibromo (Rf 0.5) products.Another 7.26 g of N-bromosuccinimide was added and the mixture stirredfor 4 additional hours. The solid was collected by vacuum filtration,washed with 20 mL of acetonitrile and dried to give a 1:1 mixture ofmono and dibromo compounds. The filtrate was concentrated andchromatographed on silica gel (ethyl acetate:hexane (1:2)) to give 1.67g of 5-bromo-4-methyl-2-oxindole as a beige solid. The remaining 1:1mixture of solids was recrystallized twice from glacial acetic acid togive 3.2 g of 5,7-dibromo-4-methyl-2-oxindole as a light orange solid.The filtrates from this material were chromatographed as above to give0.6 g of 5-bromo-4-methyl-2-oxindole and 0.5 g of5,7-dibromo-4-methyl-2-oxindole.

6-Fluoro-2-oxindole

[0196] Sodium hydride (2.6 g) and 14.5 g of dimethylmalonate was stirredand heated to 100° C. in 160 mL dimethylsulfoxide for 1.0 hour. Themixture was cooled to room temperature, 7.95 g of2,5-difluoronitrobenzene were added and mixture stirred for 30 minutes.The mixture was then heated to 100° C. for 1.0 hour, cooled to roomtemperature and poured into 400 mL of saturated ammonium chloridesolution. The mixture was extracted with 200 mL of ethyl acetate and theorganic layer washed with brine, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was crystallized from methanol togive 24.4 g (80% yield) of dimethyl 4-fluoro-2-nitrophenylmalonate as awhite solid, Rf 0.2 on thin layer chromatography (ethyl acetate:hexane1:6, silica gel). The filtrate was concentrated and chromatographed on acolumn of silica gel (ethyl acetate:hexane 1:8) to give an additional5.03 g of dimethyl 4-fluoro-2-nitro-phenylmalonate, for a total of 29.5g (96% yield).

[0197] Dimethyl 4-fluoro-2-nitrophenylmalonate (5.0 g) was refluxed in20 mL of 6N hydrochloric acid for 24 hours. The reaction was cooled andthe white solid collected by vacuum filtration, washed with water anddried to give 3.3 g (87% yield) of 4-fluoro-2-nitrophenylacetic acid, Rf0.6 on thin layer chromatography (ethyl acetate:hexane 1:2, silica gel).

[0198] 4-Fluoro-2-nitrophenylacetatic acid (3.3 g) dissolved in 15 mL ofacetic acid was hydrogenated over 0.45 g of 10% palladium on carbon at60 psi H₂ for 2 hours. The catalyst was removed by filtration and washedwith 15 mL of methanol. The combined filtrates were concentrated anddiluted with water. The precipitate was collected by vacuum filtration,washed with water and dried to give 1.6 g (70% yield) of6-fluoro-2-oxindole, Rf 0.24 on thin layer chromatography. The filtratewas concentrated to give a purple solid with an NNM spectrum similar tothe first crop. Chromatography of the purple solid (ethyl acetate:hexane1:2, silica gel) gave a second crop of 6-fluoro-2-oxindole as a whitesolid.

5-Aminosulfonyl-2-oxindole

[0199] To a 100 mL flask charged with 27 mL of chlorosulfonic acid wasadded slowly 13.3 g of 2-oxindole. The reaction temperature wasmaintained below 30° C. during the addition. After the addition, thereaction mixture was stirred at room temperature for 1.5 hr, heated to68° C. for Ihr, cooled, and poured into water. The precipitate waswashed with water and dried in a vacuum oven to give 11.0 g of5-chlorosulfonyl-2-oxindole (50% yield) which was used without furtherpurification.

[0200] 5-Chlorosulfonyl-2-oxindole (2.1 g) was added to 10 mL ofammonium hydroxide in 10 mL of ethanol and stirred at room temperatureovernight. The mixture was concentrated and the solid collected byvacuum filtration to give 0.4 g (20% yield) of the title compound as anoff-white solid.

5-Methylaminosulfonyl-2-oxindole

[0201] A suspension of 3.38 g of 5-chlorosulfonyl-2-oxindole in 10 mL 2Mmethylamine in tetrahydrofuran was stirred at room temperature for 4hours during which time a white solid formed. The precipitate wascollected by vacuum filtration, washed twice with 5 mL of water anddried under vacuum at 40° C. overnight to give 3.0 g (88% yield) of5-methylaminosulfonyl-2-oxindole.

5-(4-Trifluoromethylphenylaminosulfonyl)-2-oxindole

[0202] A suspension of 2.1 g of 5-chlorosulfonyl-2-oxindole, 1.6 g of4-trifluoromethylaniline and 1.4 g of pyridine in 20 mL ofdichloromethane was stirred at room temperature for 4 hours. Theprecipitate which formed was collected by vacuum filtration, washedtwice with 5 mL of water and dried under vacuum at 40° C. overnight togive 2.4 g of crude product containing some impurities by thin layerchromatography. The crude product was chromatographed on silica geleluting with ethyl acetate:hexane (1:2) to give 1.2 g (37% yield) of5-(4-trifluoromethylphenyl-aminosulfonyl)-2-oxindole.

5-(Morpholinosulfonyl)-2-oxindole

[0203] A suspension of 2.3 g of 5-chlorosulfonyl-2-oxindole and 2.2 g ofmorpholine in 50 mL of dichloromethane was stirred at room temperaturefor 3 hours. The white precipitate was collected by vacuum filtration,washed with ethyl acetate and hexane and dried under vacuum at 40° C.overnight to give 2.1 g (74% yield) of5-(morpholinosulfonyl)-2-oxindole.

6-Trifluoromethyl-2-oxindole

[0204] Dimethylsulfoxide (330 mL) was added to 7.9 g of sodium hydridefollowed by dropwise addition of 43.6 g diethyloxalate. The mixture washeated to 100° C. for 1.0 hour and cooled to room temperature.2-Nitro-4- trifluoromethyltoluene (31.3 g) was added, the reactionstirred for 30 minutes at room temperature and then heated to 100° C.for 1 hour. The reaction was cooled and poured into a mixture ofsaturated aqueous ammonium chloride, ethyl acetate and hexane. Theorganic layer was washed with saturated ammonium chloride, water andbrine, dried, and concentrated to give dimethyl2-(2-nitro-4-trifluoromethylphenyl)malonate.

[0205] The diester was dissolved in a mixture of 6.4 g of lithiumchloride and 2.7 mL of water in 100 mL of dimethylsulfoxide and heatedto 100° C. for 3 hours. The reaction was cooled and poured into amixture of ethyl acetate and brine. The organic phase was washed withbrine, dried with sodium sulfate, concentrated and chromatographed onsilica gel (10% ethyl acetate in hexane). The fractions containingproduct were evaporated to give 25.7 g of methyl2-nitro-4-trifluoromethylphenylacetate.

[0206] Methyl 2-nitro-4-trifluoromethylphenylacetate (26 mg) washydrogenated over 10% palladium on carbon and then heated at 100° C. for3 hours. The catalyst was removed by filtration and the solventevaporated to give the title compound.

5-(2-Chloroethyl)oxindole

[0207] 5-Chloroacetyl-2-oxindole(4.18 g) in 30 mL of trifluoroaceticacid in an ice bath was treated with 4.65 g of triethylsilane andstirred at room temperature for 3 hours. The mixture was poured into 150mL of water and the precipitate collected by vacuum filtration, washedwith 50 mL of water and dried to give 2.53 g (65% yield) of5-(2-chloroethyl)-2-oxindole as a reddish-brown solid.

5-Methoxycarbonyl-2-oxindole

[0208] 5-Iodo-2-oxindole (17 g) was refluxed with 2 g of palladiumdiacetate, 18.2 g of triethylamine, 150 mL of methanol, 15 mL ofdimethylsulfoxide and 2.6 g of DPPP in an atmosphere saturated withcarbon monoxide. After 24 hours, the reaction was filtered to remove thecatalyst and the filtrate concentrated. The concentrate waschromatographed on silica gel (30% ethyl acetate in hexane). Thefractions containing product were concentrated and allowed to stand. Theprecipitated product was collected by vacuum filtration to give 0.8 g(7%) of the title compound as an off-white solid.

4-Carboxy-2-oxindole

[0209] A solution of trimethylsilyldiazomethane in hexane (2M) was addeddropwise to a solution of 2.01 g of 2-chloro-3-carboxynitrobenzene in 20mL methanol at room temperature until no further gas evolution occurred.The excess trimethylsilyldiazo-methane was quenched with acetic acid.The reaction mixture was dried by rotary pump and the residue wasfurther dried in a vacuum oven overnight. The product(2-chloro-3-methoxycarbonylnitrobenzene) was pure enough for thefollowing reaction.

[0210] Dimethyl malonate (6.0 mL) was added to an ice-cold suspension of2.1 g of sodium hydride in 15 mL of DMSO. The reaction mixture was thenstirred at 100° C. for 1.0 h and then cooled to room temperature.2-Chloro-3-methoxycarbonyl-nitrobenzene (2.15 g) was added to the abovemixture in one portion and the mixture was heated to 100° C. for 1.5 h.The reaction mixture was then cooled to room temperature and poured intoice water, acidified to pH 5, and extracted with ethyl acetate. Theorganic layer was washed with brine, dried over anhydrous sodium sulfateand concentrated to give 3.0 g of the dimethyl2-methoxycarbonyl-6-nitrophenylmalonate.

[0211] Dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0 g) wasrefluxed in 50 mL of 6 N hydrochloric acid overnight. The mixture wasconcentrated to dryness and refluxed for 2 hours with 1.1 g of tin(II)chloride in 20 mL of ethanol. The mixture was filtered through Celite,concentrated and chromatographed on silica gel (ethylacetate:hexane:acetic acid) to give 0.65 g (37% yield) of4-carboxy-2-oxindole as a white solid.

5-Carboxy-2-oxindole

[0212] 2-Oxindole (6.7 g) was added to a stirred suspension of 23 g ofaluminum chloride in 30 mL of dichloroethane in an ice bath.Chloroacetyl chloride (11.3 g) was slowly added and hydrogen chloridegas was evolved. After ten minutes of stirring, the reaction was warmedat 40 to 50° C. for 1.5 hours. Thin layer chromatography (ethyl acetate,silica gel) showed no remaining starting material. The mixture wascooled to room temperature and poured into ice water. The precipitatewas collected by vacuum filtration, washed with water and dried undervacuum to give 10.3 g (98%) of 5-chloroacetyl-2-oxindole as an off-whitesolid.

[0213] A suspension of 9.3 g of 5-chloroacetyl-2-oxindole was stirred in90 mL pyridine at 80 to 90° C. for 3 hours then cooled to roomtemperature. The precipitate was collected by vacuum filtration andwashed with 20 mL ethanol. The solid was dissolved in 90 mL 2.5N sodiumhydroxide and stirred at 70 to 80° C. for 3 hours. The mixture wascooled to room temperature and acidified to pH 2 with 0.5 N hydrochloricacid. The precipitate was collected by vacuum filtration and washedthoroughly with water to give crude 5-carboxy-2-oxindole as a dark brownsolid. After standing overnight the filtrate yielded 2 g of5-carboxy-2-oxindole as a yellow solid. The crude dark brown product wasdissolved in hot methanol, the insoluble material removed by filtrationand the filtrate concentrated to give 5.6 g of 5-carboxy-2-oxindole as abrown solid. The combined yield was 97%.

5-Carboxyethyl-2-oxindole

[0214] 5-Cyanoethyl-2-oxindole (4.02 g) in 10 mL of water containing 25mL of concentrated hydrochloric acid was refluxed for 4 hours. Themixture was cooled, water added and the resulting solid collected byvacuum filtration, washed with water and dried to give 1.9 g (44% yield)of the title compound as a yellow solid.

5-Iodo-4-methyl-2-oxindole

[0215] To 2 g of 4-methyl-2-oxindole in 40 mL of glacial acetic acid inan ice bath was added 3.67 g N-iodosuccinimide. The mixture was stirredfor 1 hour, diluted with 100 mL 50% acetic acid in water and filtered.The resulting white solid was dried under high vacuum to give 3.27 g(88% yield) of the title compound as an off-white solid.

5-Chloro-4-methyl-2-oxindole

[0216] A suspension of 3.0 g of 4-methyl-2-oxindole was stirred in 50 mLof acetonitrile at room temperature while 3.3 g of N-chlorosuccinimidewas added in portions. Trifluoroacetic acid (1 mL) was then added. Thesuspension was stirred at room temperature for 3 days during which timesolid was always present. The white solid was collected by vacuumfiltration, washed with a small amount of cold acetone and driedovernight in a vacuum oven at 40° C. to give 2.5 g (68%) of5-chloro-4-methyl-2-oxindole.

5-Butyl-2-oxindole

[0217] Triethylsilane (2.3 g) was added to 2 g 4-butanoyl-2-oxindole in20 mL of trifluoroacetic acid at room temperature and the solutionstirred for 3 hours. The reaction was poured into ice water to give ared oil which solidified after standing. The solid was collected byvacuum filtration, washed with water and hexane and dried to give 1.7 g(91% yield) of the title compound as an off-white solid.

5-Ethyl-2-oxindole

[0218] To 5-Acetyl-2-oxindole (2 g) in 15 mL of trifluoroacetic acid inan ice bath was slowly added 1.8 g of triethylsilane; the reaction wasthen stirred at room temperature for 5 hours. One mL of triethylsilanewas added and the stirring continued overnight. The reaction mixture waspoured into ice water and the resulting precipitate collected by vacuumfiltration, washed copiously with water and dried under vacuum to give1.3 g (71% yield) of the title compound as a yellow solid.

5-(Morpholin-4-ethyl)-2-oxindole

[0219] 5-Chloroethyl-2-oxindole (2.3 g), 1.2 mL of morpholine and 1.2 mLof diisopropylethylamine were heated overnight at 100° C. in 10 mL ofdimethylsulfoxide. The mixture ws cooled, poured into water and extactedwith ethyl acetate. The organic layer was washed with brine, dried andevaporated. The residue was chromatographed on silica gel (5% methanolin chloroform) to give 0.9 g (31%) of the title compound as a whitesolid.

5-(4-Methoxycarbonylbenzamido)-2-oxindole

[0220] A mixture of 82.0 mg 5-amino-2-oxindole and 131.0 mg4-methoxycarbonylbenzoyl chloride in pyridine was stirred at roomtemperature for 3 hr and poured into ice water. The precipitate wasfiltered, washed with water and dried in a vacuum oven to give 138.0 mgof 5-(4-methoxycarbonylbenzamido)-2-oxindole (81% yield).

5-(4-Carboxybenzamido)-2-oxindole

[0221] 5-(4-Methoxycarbonylbenzamido)-2-oxindole (0.9 g) and 0.4 g ofsodium hydroxide in 25 mL of methanol were refluxed for 3 hours. Themixture was concentrated, water added, and the mixture acidified with 6Nhydrochloric acid. The precipitate was collected by vacuum filtration togive 0.75 g (87%) of the title compound as a white solid.

5-Methoxy-2-oxindole

[0222] Chloral hydrate (9.6 g) was dissolved in 200 mL of watercontaining 83 g of sodium sulfate. The solution was warmed to 60° C., asolution of 11.4 g of hydroxylamine hydrochloride in 50 mL of water wasadded and the mixture was held at 60° C. In a separate flask, 6.4 g of4-anisidine and 4.3 mL of concentrated hydrochloric acid in 80 mL ofwater was warmed to 80° C. The first solution was added to the secondand the mixture refluxed for 2 minutes after which it was cooled slowlyto room temperature and then cooled in an ice bath. The tan precipitatewas collected by vacuum filtration, washed with water and dried undervacuum to give 8.6 g ( 85% yield) of N-(2-hydroximino-acetyl)anisidine.

[0223] Concentrated sulfuric acid (45 mL) containing 5 mL of water waswarmed to 60° C. and 8.6 g of N-(2-hydroximinoacetyl)anisidine was addedin one portion. The stirred mixture was heated to 93° C. for 10 minutesand then allowed to cool to room temperature. The mixture was pouredinto 500 g of ice and extracted 3 times with ethyl acetate. The combinedextracts were dried over anhydrous sodium sulfate and concentrated togive 5.1 g (65% yield) of 5-methoxyisatin as a dark red solid.5-Methoxyisatin (5.0 g) and 30 mL of hydrazine hydrate were heated toreflux for 15 minutes. The reaction mixture was cooled to roomtemperature and 50 mL of water was added. The mixture was extracted 3times with 25 mL of ethyl acetate each time, the organic layerscombined, dried over anhydrous sodium sulfate and concentrated to give ayellow solid. The solid was stirred in ethyl acetate and 1.1 g ofinsoluble material was removed by vacuum filtration and saved. Thismaterial proved to be 2-hydrazinocarbonylmethyl-4-anisidine. Thefiltrate was concentrated and chromatographed on silica gel eluting withethyl acetate:hexane (1:1) to give 0.7 g of 5-methoxy-2-oxindole as ayellow solid. The 1.1 g of 2-hydrazino-carbonylmethyl-4-anisidine wasrefluxed for 1 hour in 20 mL of 1N sodium hydroxide. The mixture wascooled, acidified to pH 2 with concentrated hydrochloric acid andextracted 3 times with 25 mL of ethyl acetate each time. The organicextracts were combined, washed with brine, dried over anhydrous sodiumsulfate and concentrated to give 0.8 g of 5-methoxy-2-oxindole as ayellow solid. The combined yield was 1.5 g or 33%.

7-Azaoxindole

[0224] 3,3-Dibromo-7-azaoxindole (2.9 g) was dissolved in a mixture of20 mL of acetic acid and 30 mL of acetonitrile. To the solution wasadded 6.5 g of zinc dust. The mixture was stirred for 2 hrs at roomtemperature. The solid was filtered from the mixture and the solventevaporated. The residue was slurried with ethyl acetate. The ethylacetate solution containing insoluble solid was passed through a shortcolumn of silica gel. The collected ethyl acetate solution wasevaporated and the residue dried under vacuum to give 1.8 g (yield 91%)of 7-azaoxindole acetic acid salt.

5-Dimethylaminosulfonyl-2-oxindole

[0225] A suspension of 2.3 g 5-chlorosulfonyl-2-oxindole in 10 mL 2Mdimethylamine in methanol was stirred at room temperature for 4 hours atwhich time a white solid formed. The precipitate was collected by vacuumfiltration, washed with 5 mL of 1N sodium hydroxide and 5 mL of 1Nhydrochloric acid and dried under vacuum at 40° C. overnight to give 1.9g (79% yield) of 5-dimethylamino-sulfonyl-2-oxindole.

6-Phenyl-2-oxindole

[0226] Dimethyl malonate (10 mL) in 25 mL of dimethylsulfoxide was addeddropwise to 3.5 g sodium hydride suspended in 25 mL dimethylsulfoxideand the mixture heated at 100° C. for 10 minutes. The mixture was cooledto room temperature and 4.7 g of 4-fluoro-3-nitrobiphenyl in 25 mLdimethylsulfoxide was added. The mixture was heated at 100° C. for 2hours, cooled and quenched with 300 mL of saturated ammonium chloridesolution. The mixture was extracted three times with ethyl acetate andthe combined organic layers washed with water and brine and evaporatedto give, as a yellow oil, crude dimethyl-3-nitrobiphenyl-4-malonate.

[0227] Crude dimethyl-3-nitrobiphenyl-4-malonate was refluxed in 30 mLof 6 N hydrochloric acid for 24 hours. The precipitate was collected byfiltration, washed with water and dried to give 4.5 g of3-nitrobiphenyl-4-acetic acid as a cream colored solid.

[0228] Iron powder (2.6 g) was added all at once to 4.5 g of3-nitrobiphenyl-4-acetic acid in 40 mL of acetic acid. The mixture wasrefluxed for 2 hours, concentrated to dryness and taken up in ethylacetate. The solids were removed by filtration and the filtrate washedtwice with 1N hydrochloric acid and brine and dried over anhydroussodium sulfate. The filtrate was concentrated to give 3.4 g (93% yield)of 6-phenyl-2-oxindole as a light brown solid.

6-(2-Methoxyphenyl)-2-oxindole

[0229] Tetrakis(triphenylphosphine)palladium (I g) was added to amixture of 5 g 2-methoxyphenylboronic acid, 6.6 g5-bromo-2-fluoronitrobenzene and 30 mL of 2 M sodium carbonate solutionin 50 mL of toluene and 50 mL of ethanol. The mixture was refluxed for 2hours, concentrated, and the residue extracted twice with ethyl acetate.The ethyl acetate layer was washed with water and brine, then dried, andconcentrated to give a dark green oil which solidified on standing,crude 4-fluoro-2′-methoxy-3-nitrobiphenyl.

[0230] Dimethyl malonate (14 mL) was added dropwise to 2.9 g of sodiumhydride suspended in 50 mL of dimethylsulfoxide. The mixture was heatedat 100° C. for 15 minutes and cooled to room temperature. Crude4-fluoro-2′-methoxy-3-nitrobiphenyl in 60 mL of dimethylsulfoxide wasadded and the mixture was heated at 100° C. for 2 hours. The reactionmixture was cooled and quenched with 300 mL of saturated sodium chloridesolution and extracted twice with ethyl acetate. The extracts werecombined, washed with saturated ammonium chloride, water and brine,dried over anhydrous sodium sulfate and concentrated to give crudedimethyl 2′-methoxy-3-nitrobiphenyl-4-malonate as a yellow oil.

[0231] Crude dimethyl 2′-methoxy-3-nitrobiphenyl-4-malonate was heatedat 100° C. in 50 mL of 6 N hydrochloric acid for 24 hours and cooled.The precipitate was collected by filtration, washed with water andhexane, and dried to give 9.8 of 2′-methoxy-2-nitrobiphenyl-4acetic acidas a light tan solid.

[0232] Iron powder (5 g) was added in one portion to 9.8 g of2′-methoxy-3-nitrobiphenyl-4-acetic acid in 50 mL of glacial acetic acidwas heated to 100° C. for 3 hours. The reaction mixture was concentratedto dryness, sonicated in ethyl acetate and filtered to remove theinsolubles. The filtrate was washed twice with 1N hydrochloric acid,water and then brine, dried over anhydrous sodium sulfate andconcentrated. The residue was chromatographed on silica gel in ethylacetate:hexane (1:2) to give 5.4 g of 6-(2-methoxyphenyl)-2-oxindole asa rose colored solid.

6-(3-Methoxyphenyl)-2-oxindole

[0233] Tetrakis(triphenylphosphine)palladium (0.8 g) was added to amixture of 5 g 3-methoxyphenylboronic acid, 5 g5-bromo-2-fluoro-nitrobenzene and 11 mL of 2 M sodium carbonate solutionin 100 mL of toluene. The mixture was refluxed for 2 hours, diluted withwater and extracted with ethyl acetate. The ethyl acetate was washedwith saturated sodium bicarbonate and brine and then dried andconcentrated to give an oily solid. The solid was chromatographed onsilica gel (ethyl acetate:hexane (1:6)) to give 4.3 g (77% yield) of4-fluoro-3′-methoxy-3- nitrobiphenyl.

[0234] Dimethyl malonate (9.7 mL) was added dropwise to 2.0 g sodiumhydride suspended in 50 mL dimethylsulfoxide. The mixture was heated to100° C. for 35 minutes and cooled to room temperature.4-Fluoro-2′-methoxy-3-nitrobiphenyl (4.2 g) in 50 mL dimethylsulfoxidewas added and the mixture was heated at 100° C. for 1 hour. The reactionmixture was cooled and quenched with 300 mL of saturated ammoniumchloride solution and extracted twice with ethyl acetate. The extractswere combined, washed with brine, dried over anhydrous sodium sulfateand concentrated to give crude dimethyl3′-methoxy-3-nitrobiphenyl-4-malonate as a pale yellow solid.

[0235] Crude dimethyl 3′-methoxy-3-nitro-biphenyl-4-malonate was heatedat 110° C. in 45 mL 6N hydrochloric acid for 4 days and then cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 5.3 g of 3′-methoxy-2-nitrobiphenyl-4-acetic acid as alight tan solid.

[0236] 3′-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 g) was dissolved inmethanol and hydrogenated over 0.8 g of 10% palladium on carbon for 3hours at room temperature. The catalyst was removed by filtration,washed with methanol and the filtrates combined and concentrated to givea brown solid. The solid was chromatographed on silica gel in ethylacetate:hexane:acetic acid (33:66:1) to give 3.0 g of6-(3-methoxypheny)-2-oxindole as a pink solid.

6-(4-Methoxyphenyl)-2-oxindole

[0237] Tetrakis(triphenylphosphine)palladium (I g) was added to amixture of 5 g of 4methoxyphenylboronic acid, 6.6 g of5-bromo-2-fluoronitrobenzene and 30 mL of 2 M sodium carbonate solutionin 50 mL of toluene and 50 mL of ethanol. The mixture was refluxed for 2hours, concentrated, and the residue extracted twice with ethyl acetate.The ethyl acetate layer was washed with water and brine, dried, andconcentrated to give a brown oily solid. The solid was chromatographedon silica gel (5% ethyl acetate in hexane) to give crude4-fluoro-4′-methoxy-3-nitrobiphenyl as a pale yellow solid.

[0238] Dimethyl malonate (10 mL) was added dropwise to 2.0 g of sodiumhydride suspended in 60 mL of dimethylsulfoxide. The mixture was heatedto 100° C. for 10 minutes and cooled to room temperature. Crude4-fluoro-2′-methoxy-3-nitrobiphenyl (5.2 g) in 50 mL dimethylsulfoxidewas added and the mixture was heated at 100° C. for 2 hours. Thereaction mixture was cooled and quenched with 300 mL of saturated sodiumchloride solution and extracted three times with ethyl acetate. Theextracts were combined, washed with saturated ammonium chloride, waterand brine, dried over anhydrous sodium sulfate and concentrated to givecrude dimethyl 4′-methoxy-3-nitrobiphenyl-4malonate as a yellow oil.

[0239] Crude dimethyl 4′-methoxy-3-nitro-biphenyl-4-malonate was heatedat 100° C. in 60 mL of 6N hydrochloric acid for 15 hours and cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 7.2 g of crude 4′-methoxy-3-nitrobiphenyl-4-aceticacid as a light tan solid.

[0240] Iron powder (3.6 g) was added in one portion to 7.2 g of4′-methoxy-3-nitrobiphenyl-4-acetic acid in 50 mL of glacial acetic acidand heated at 100° C. overnight. The reaction mixture was concentratedto dryness, sonicated in ethyl acetate and filtered to remove theinsolubles. The filtrate was washed twice with 1N hydrochloric acid andbrine, dried over anhydrous sodium sulfate and concentrated to give 2.7g of 6-(4-methoxyphenyl)-2-oxindole as a rose colored solid.

6- (3-Ethoxyphenyl)-2-oxindole

[0241] Tetrakis(triphenylphosphine)palladium (0.8 g) was added to amixture of 4.2 g of 3-ethoxyphenylboronic acid, 5.0 g of5-bromo-2-fluoronitrobenzene and 22 mL of 2 M sodium carbonate solutionin 50 mL of toluene and 50 mL of ethanol. The mixture was refluxed for 2hours, concentrated, water was added and the mixture was extracted twicewith ethyl acetate. The ethyl acetate layer was washed with water andbrine, then dried, and concentrated. The residue was chromatographed onsilica gel (5% ethyl acetate in hexane) to give 5.3 g (90% yield) ofcrude 4-fluoro-3′-ethoxy-3-nitrobiphenyl as a yellow oil.

[0242] Dimethyl malonate (11.4 mL) was added dropwise to 4.0 g sodiumhydride suspended in 20 mL dimethylsulfoxide. The mixture was heated to100° C. for 10 minutes and then cooled to room temperature. Crude4-fluoro-3′-ethoxy-3-nitrobiphenyl (5.3 g) in 25 mL of dimethylsulfoxidewas added and the mixture was heated at 100° C. for 2 hours. Thereaction mixture was cooled and quenched with 300 mL of saturatedammonium chloride solution and extracted three times with ethyl acetate.The extracts were combined, washed with water and brine and then driedover anhydrous sodium sulfate and concentrated to give crude dimethyl3′-ethoxy-3-nitrobiphenyl-4-malonate as a yellow oil.

[0243] Crude dimethyl 3′-ethoxy-3-nitrobiphenyl-4-malonate was heated at100° C. in 60 mL of 6N hydrochloric acid for 4 days and then cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 4.7 g of crude 3′-ethoxy-3-nitrobiphenyl-4-acetic acidas a light tan solid.

[0244] Iron powder (2.4 g) was added in one portion to 4.6 g of3′-ethoxy-3-nitrobiphenyl-4-acetic acid in 40 mL of glacial acetic acidand refluxed for 2 hours. The reaction mixture was concentrated todryness, treated repeatedly with ethyl acetate and filtered to removethe insolubles. The filtrate was washed twice with 1N hydrochloric acidand brine and then dried over anhydrous sodium sulfate and concentratedto give 3.5 g (91% yield) of 6-(3-ethoxyphenyl)-2-oxindole as a lightbrown solid.

6-Bromo-2-oxindole

[0245] Dimethyl malonate (13 mL) was added dropwise to 2.7 g sodiumhydride suspended in 20 mL dimethylsulfoxide. The mixture was heated to100° C. for 10 minutes and then cooled to room temperature.5-Bromo-2-fluoronitrobenzene (5.0 g) in 25 mL of dimethylsulfoxide wasadded and the mixture was heated at 100° C. for 2 hours. The reactionmixture was cooled and quenched with 300 mL of saturated ammoniumchloride solution and extracted three times with ethyl acetate. Theextracts were combined, washed with saturated ammonium chloride, waterand brine, dried over anhydrous sodium sulfate and concentrated to givecrude dimethyl 4-bromo-2-nitrophenylmalonate as a pale yellow oil.

[0246] Crude dimethyl 4-bromo-2-nitrophenylmalonate was heated at 110°C. in 40 mL of 6N hydrochloric acid for 24 hours and then cooled. Theprecipitate was collected by filtration, washed with water and dried togive 5.3 g (89% yield) of 4-bromo-2-nitro-phenylacetic acid as an offwhite solid.

[0247] 4-Bromo-2-nitrophenylacetic acid (0.26 g), 0.26 g zinc powder and3 mL 50% sulfuric acid in 5 mL of ethanol were heated at 100° C.overnight. The reaction mixture was filtered, diluted with a littleacetic acid, concentrated to remove ethanol, diluted with water andextracted twice with ethyl acetate. The combined extracts were washedwith brine, dried over anhydrous sodium sulfate and concentrated to give0.19 g (90% yield) of 6-bromo-2-oxindole as a yellow solid.

5-Acetyl-2-oxindole

[0248] 2-Oxindole (3 g) was suspended in 1,2-dichloroethane and 3.2 mLacetyl chloride were slowly added. The resulting suspension was heatedto 50° C. for 5 hours, cooled, and poured into water. The resultingprecipitate was collected by vacuum filtration, washed copiously withwater and dried under vacuum to give 2.9 g (73% yield) of the titlecompound as a brown solid.

5-Butanoyl-2-oxindole

[0249] To 15 g aluminum chloride suspended in 30 mL 1,2-dichloro-ethanein an ice bath was added 7.5 g of 2-oxindole and then 12 g of butanoylchloride. The resulting suspension was heated to 50° C. overnight. Themixture was poured into ice water and extracted 3 times with ethylacetate. The combined ethyl acetate layers were washed with brine, driedover sodium sulfate, and concentrated to dryness to give a brown solid.The solid was chromatographed on silica gel (50% ethyl acetate inhexane) to give 3 g (25%) of the title compound as a yellow solid.

5-Cyanoethyl-2-oxindole

[0250] Potassium cyanide (2.0 g) was added to 15 mL ofdimethyl-sulfoxide and heated to 90° C. 5-Chloroethyl-2-oxindole (3.0 g)dissolved in 5 mL dimethyl sulfoxide was added slowly with stirring, andthe reaction heated to 150° C. for 2 hours. The mixture was cooled,poured into ice water and the precipitate collected by vacuumfiltration, washed with water, dried and then chromatographed on silicagel (5% methanol in chloroform) to give 1.2 g (42% yield) of the titlecompound.

6-Morpholin-4-yl)-2-oxindole

[0251] 6-Amino-2-oxindole (2.2 g), 4.0 g 2, 2′-dibromoethyl ether and7.9 g sodium carbonate were refluxed in 20 ml ethanol overnight,concentrated and diluted with 50 ml of water. The mixture was extractedthree times with 50 ml of ethyl acetate and the organic extractscombined, washed with 20 ml of brine, dried over anhydrous sodiumsulfate and concentrated to dryness. The solid was chromatographed on acolumn of silica gel (ethyl acetate:hexane (1:1) containing 0.7% aceticacid) to give 1.2 g (37% yield) of the title compound as a beige solid.

6-(3-Trifluoroacetylphenyl)-2-oxindole

[0252] 3-Aminophenylboronic acid (3.9 g), 5 g5-bromo-2-fluoro-nitrobenzene, 0.8 gtetrakis(triphenylphosphine)palladium and 23 mL of 2 M sodiumbicarbonate solution in 50 mL of toluene were refluxed under nitrogenfor 2.5 hours. The reaction mixture was poured into 200 mL of ice waterand the mixture extracted three times with 50 mL of ethyl acetate. Thecombined organic layers were washed with 50 mL of water and 20 mL ofbrine, dried over, anhydrous sodium sulfate and concentrated to give 9.7g (92% yield) of 2-fluoro-5-(3-aminophenyl)nitrobenzene as a dark brownoil.

[0253] Trifluoroacetic anhydride (5.4 mL) was slowly added to a stirredsolution of 9.7 g 2-fluoro-5-(3-aminophenyl)-nitrobenzene and 5.3 mL oftriethylamine in 50 mL of dichloromethane at 0° C. and the mixture wasstirred for an additional 20 minutes. The mixture was concentrated andthe residue chromatographed on a column of silica gel (10% ethyl acetatein hexane) to give 8.6 g (65% yield) of2-fluoro-5-(3-trifluoroacetamidophenyl)nitrobenzene as a pale orange oilwhich solidified on standing.

[0254] Dimethyl malonate (9.6 mL) was added dropwise to a stirredsuspension of 3.2 g of 60% sodium hydride in mineral oil in 40 mLanhydrous dimethylsulfoxide under nitrogen. The mixture was stirred for10 minutes and 2-fluoro-5-(3-trifluoroacetamidophenyl)nitrobenzene in 20mL dimethylsulfoxide was added. The resulting dark red mixture washeated to 100° C. for 2 hours. The reaction was quenched by pouring into100 mL of saturated ammnonium chloride solution and extracted twice with50 mL of ethyl acetate. The organic phase was washed with 50 mL each ofsaturated ammonium chloride solution, water, and brine, dried overanhydrous sodium sulfate and concentrated to a yellow oil. The oil waschromatographed on a column of silica gel (ethyl acetate:hexane (1:4))to give 4.4 g (50% yield) of dimethyl2-[2-nitro-4-(3-trifluoroacetamidophenyl)phenyl] malonate as a paleyellow solid.

[0255] Dimethyl2-[2-nitro-4-(3-trifluoroacetamidophenyl)phenyl]-malonate (4.4 g) wasrefluxed overnight in 50 mL 6N hydrochloric acid. The reaction mixturewas cooled to room temperature and the solids were collected by vacuumfiltration, washed with water, and dried under vacuum to give 2.7 g (73%yield) of 2-[2-nitro-4-(3-trifluoroacetamidophenyl)phenyl] acetic acid.

[0256] 2-[2-Nitro-4-(3-trifluoroacetamidophenyl)phenyl]acetic acid (100mg) and 50 mg iron powder in 3 mL acetic acid was heated at 100° C. for2 hours. The reaction mixture was concentrated and the residue sonicatedin 5 mL ethyl acetate. The insoluble solids were removed by vacuumfiltration and the filtrate washed with 1N hydrochloric acid, water andbrine, dried over anhydrous sodium sulfate and concentrated to give 10mg (14% yield) of the title compound as a rose-colored solid.

[0257] 5. Biological Evaluation

[0258] It will be appreciated that, in any given series of compounds, aspectrum of biological activity will be afforded. In its most preferredembodiments, this invention relates to novel3-heteroarylidenyl-2-indolinones having improved hydrosolubility anddemonstrating the ability to modulate RTK and CTK activity. Thefollowing assays are employed to select those compounds demonstratingthe optimal degree of the desired activity.

[0259] As used herein, the phrase “optimal degree of the desiredactivity” refers to the lowest IC50, defined elsewhere herein, against aPTK related to a particular disorder so as to provide an organism,preferably a human, with a therapeutically effective amount of acompound of this invention at the lowest possible dosage.

[0260] B. Assay Procedures.

[0261] The following in vitro assays may be used to determine the levelof activity and effect of the different compounds of the presentinvention on one or more of the RTKs. Similar assays can be designedalong the same lines for any PTK using techniques well known in the art.

[0262] The cellular/catalytic assays described herein are performed inan ELISA format. The general procedure is as follows: a compound isintroduced to cells expressing the test kinase, either naturally orrecombinantly, for some period of time after which, if the test kinaseis a receptor, a ligand known to activate the receptor's activity isadded. The cells are lysed and the lysate is transferred to the wells ofan ELISA plate previously coated with a specific antibody recognizingthe substrate of the enzymatic phosphorylation reaction. Non-substratecomponents of the cell lysate are washed away and the amount ofphosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

[0263] The cellular/biologic assays described herein measure the amountof DNA made in response to activation of a test kinase, which is ageneral measure of a proliferative response. The general procedure forthis assay is as follows: a compound is introduced to cells expressingthe test kinase, either naturally or recombinantly, for some period oftime after which, if the test kinase is a receptor, a ligand known toactivate the receptor's activity is added. After incubation at leastovernight, a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or3H-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

[0264] 1. Cellular/Catalytic Assays

[0265] Enzyme linked immunosorbent assays (ELISA) may be used to detectand measure the presence of PTK activity. The ELISA may be conductedaccording to known protocols which are described in, for example,Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual ofClinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am.Soc. Of Microbiology, Washington, D.C.

[0266] The disclosed protocol may be adapted for determining activitywith respect to a specific RTK. For example, the preferred protocols forconducting the ELISA experiments for specific RTKs is provided below.Adaptation of these protocols for determining a compound's activity forother members of the RTK family, as well as for CTKs, is well within thescope of knowledge of those skilled in the art.

[0267] a. FLK-1

[0268] An ELISA assay is conducted to measure the kinase activity of theFLK-1 receptor and more specifically, the inhibition or activation of TKactivity on the FLK-1 receptor. Specifically, the following assay can beconducted to measure kinase activity of the FLK-1 receptor in cellsgenetically engineered to express Flk-1.

Materials And Methods.

[0269] Materials. The following reagents and supplies are used:

[0270] a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);

[0271] b. Cappel goat anti-rabbit IgG (catalog no. 55641);

[0272] c. PBS (Gibco Catalog No. 450-1300EB);

[0273] d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1%Tween-20);

[0274] e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4°C.);

[0275] f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2%Triton X-100, and 10% glycerol);

[0276] g. EDTA (0.5 M (pH 7.0) as a 100×stock);

[0277] h. Sodium orthovanadate (0.5 M as a 100×stock);

[0278] i. Sodium pyrophosphate (0.2 M as a 100×stock);

[0279] j. NUNC 96 well V bottom polypropylene plates (Applied ScientificCatalog No. AS-72092);

[0280] k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);

[0281] l. DMEM with 1X high glucose L-Glutamine (catalog No. 11965-050);

[0282] m. FBS, Gibco (catalog no. 16000-028);

[0283] n. L-glutamine, Gibco (catalog no. 25030-016);

[0284] o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 μg/100 μlstock in Milli-Q dH₂O and stored at −20° C.;

[0285] p. Affinity purified anti-FLK-l antiserum;

[0286] q. UB40 monoclonal antibody specific for phosphotyrosine (see,Fendley, et al., 1990, Cancer Research 50:1550-1558);

[0287] r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no.172-1011);

[0288] s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS)solution (100 mM citric acid (anhydrous), 250 mM Na₂HPO₄ (pH 4.0), 0.5mg/ml ABTS (Sigma catalog no. A-1888)), solution should be stored indark at 4° C. until ready for use;

[0289] t. H₂O₂ (30% solution) (Fisher catalog no. H325)

[0290] u. ABTS/H₂O (15 ml ABTS solution, 2 μl H₂O₂) prepared 5 minutesbefore use and left at room temperature;

[0291] v. 0.2 M HCl stock in H₂O;

[0292] w. dimethylsulfoxide (100%)(Sigma Catalog No. D-8418); and

[0293] y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).

[0294] Protocol. The following protocol can be used for conducting theassay:

[0295] 1. Coat Corning 96-well ELISA plates with 1.0 μg per well CappelAnti-rabbit IgG antibody in 0.1M Na₂CO₃ pH 9.6. Bring final volume to150 μl per well. Coat plates overnight at 4° C. Plates can be kept up totwo weeks when stored at 4° C.

[0296] 2. Grow cells in Growth media(DMEM, supplemented with 2.0 mML-Glutamine, 10% FBS) in suitable culture dishes until confluent at 37°C., 5% CO₂.

[0297] 3. Harvest cells by trypsinization and seed in Corning 25850polystyrene 96-well round bottom cell plates, 25.000 cells/well in 200μl of growth media.

[0298] 4. Grow cells at least one day at 37° C., 5% CO₂.

[0299] 5. Wash cells with D-PBS 1X.

[0300] 6. Add 200 μl/well of starvation media (DMEM, 2.0 mM 1-Glutamine,0.1% FBS). Incubate overnight at 37° C., 5% CO₂.

[0301] 7. Dilute Compounds 1:20 in polypropylene 96 well plates usingstarvation media. Dilute dimethylsulfoxide 1:20 for use in controlwells.

[0302] 8. Remove starvation media from 96 well cell culture plates andadd 162 μl of fresh starvation media to each well.

[0303] 9. Add 18 μl of 1:20 diluted Compound dilution (from step 7) toeach well plus the 1:20 dimethylsulfoxide dilution to the control wells(+/− VEGF), for a final dilution of 1:200 after cell stimulation. Finaldimethylsulfoxide is 0.5%. Incubate the plate at 37° C., 5% CO₂ for twohours.

[0304] 10. Remove unbound antibody from ELISA plates by inverting plateto remove liquid. Wash 3 times with TBSW+0.5% ethanolamine, pH 7.0. Patthe plate on a paper towel to remove excess liquid and bubbles.

[0305] 11. Block plates with TBSW+0.5% Ethanolamine, pH 7.0, 150 μl perwell. Incubate plate thirty minutes while shaking on a microtiter plateshaker.

[0306] 12. Wash plate 3 times as described in step 10.

[0307]13. Add 0.5 μg/well affinity purified anti-FLU-1 polyclonal rabbitantiserum. Bring final volume to 150 μl/well with TBSW+0.5% ethanolaminepH 7.0. Incubate plate for thirty minutes while shaking.

[0308] 14. Add 180 μl starvation medium to the cells and stimulate cellswith 20 μl/well 10.0 mM sodium ortho vanadate and 500 ng/ml VEGF(resulting in a final concentration of 1.0 mM sodium ortho vanadate and50 ng/ml VEGF per well) for eight minutes at 37° C., 5% CO₂. Negativecontrol wells receive only starvation medium.

[0309] 15. After eight minutes, media should be removed from the cellsand washed one time with 200 μl/well PBS.

[0310] 16. Lyse cells in 150 μl/well HNTG while shaking at roomtemperature for five minutes. HNTG formulation includes sodium orthovanadate, sodium pyrophosphate and EDTA.

[0311] 17. Wash ELISA plate three times as described in step 10.

[0312] 18. Transfer cell lysates from the cell plate to ELISA plate andincubate while shaking for two hours. To transfer cell lysate pipette upand down while scrapping the wells.

[0313] 19. Wash plate three times as described in step 10.

[0314]20. Incubate ELISA plate with 0.02 μg/well UB40 in TBSW+05%ethanolamine. Bring final volume to 150 μl/well. Incubate while shakingfor 30 minutes.

[0315] 21. Wash plate three times as described in step 10.

[0316] 22. Incubate ELISA plate with 1:10,000 diluted EIA grade goatanti-mouse IgG conjugated horseradish peroxidase in TBSW+0.5%ethanolamine, pH 7.0. Bring final volume to 150 μl/well. Incubate whileshaking for thirty minutes.

[0317] 23. Wash plate as described in step 10.

[0318] 24. Add 100 μl of ABTS/H₂O₂ solution to well. Incubate tenminutes while shaking.

[0319] 25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final to stop the colordevelopment reaction. Shake 1 minute at room temperature. Remove bubbleswith slow stream of air and read the ELISA plate in an ELISA platereader at 410 nm.

[0320] b. HER-2 ELISA

Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells

[0321] HER2 kinase activity in whole EGFR-NIH3T3 cells are measured asdescribed below:

[0322] Materials and Reagents. The following materials and reagents canbe used to conduct the assay:

[0323] a. EGF: stock concentration: 16.5 ILM; EGF 201, TOYOBO, Co., Ltd.Japan.

[0324] b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFRextracellular domain).

[0325] c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see,Fendley, et al., supra).

[0326] d. Detection antibody: Goat anti-rabbit lgG horse radishperoxidase conjugate, TAGO, Inc., Burlingame, Calif. Tris-HCl, pH 7.2 50mM NaCl 150 mM Triton X-100 0.1 f. HNTG 5X stock: HEPES 0.1 M NaCl 0.75M Glycerol 50% Triton X-100 1.0% g. ABTS stock: Citric Acid 100 mMNa₂HPO₄ 250 mM HCl, conc. 0.5 pM ABTS 0.5 mg/ml

[0327] h. Stock reagents of:

[0328] EDTA 100 mM pH 7.0

[0329] Na₃VO₄ 0.5 M

[0330] Na₄(P₂O₇) 0.2 M

[0331] Procedure. The following protocol is used:

[0332] A. Pre-coat ELISA Plate

[0333] 1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with05-101 antibody at 0.5 g per well in PBS, 100 μl final volume/well, andstore overnight at 4° C. Coated plates are good for up to 10 days whenstored at 4° C.

[0334] 2. On day of use, remove coating buffer and replace with 100 μlblocking buffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubatethe plate, shaking, at room temperature (about 23° C. to 25° C.) for 30minutes. Just prior to use, remove blocking buffer and wash plate 4times with TBST buffer.

[0335] B. Seeding Cells

[0336] 1. An NIH3T3 cell line overexpressing a chimeric receptorcontaining the EGFR extracellular domain and intracellular HER2 kinasedomain can be used for this assay.

[0337] 2. Choose dishes having 80-90% confluence for the experiment.Trypsinize cells and stop reaction by adding 10% fetal bovine serum.Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at1500 rpm, at room temperature for 5 minutes.

[0338] 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum),and count the cells using trypan blue. Viability above 90% isacceptable. Seed cells in DMEM medium (0.5% bovine serum) at a densityof 10,000 cells per well, 100 μl per well, in a 96 well microtiterplate. Incubate seeded cells in 5% CO₂ at 37° C. for about 40 hours.

[0339] C. Assay Procedures

[0340] 1. Check seeded cells for contamination using an invertedmicroscope. Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium,then transfer 5 μl to a TBST well for a final drug dilution of 1:200 anda final DMSO concentration of 1%. Control wells receive DMSO alone.Incubate in 5% CO₂ at 37° C. for two hours.

[0341] 2. Prepare EGF ligand: dilute stock EGF in DMEM so that upontransfer of 10 μl dilute EGF (1:12 dilution), 100 nM final concentrationis attained.

[0342] 3. Prepare fresh HNTG* sufficient for 100 μl per well; and placeon ice. HNTG stock 2.0 ml milli-Q H₂O 7.3 ml EDTA, 100 mM, pH 7.0 0.5 mlNa₃VO₄, 0.5 M 0.1 ml Na₄(P₂O₇), 0.2 M 0.1 ml

[0343] 4. After 120 minutes incubation with drug, add prepared SGFligand to cells, 10 μl per well, to a final concentration of 100 nM.Control wells receive DMEM alone. Incubate, shaking, at roomtemperature, for 5 minutes.

[0344] 5. Remove drug, EGF, and DMEM. Wash cells twice with PBS.Transfer HNTG to cells, 100 μl per well. Place on ice for 5 minutes.Meanwhile, remove blocking buffer from other ELISA plate and wash withTBST as described above.

[0345] 6. With a pipette tip securely fitted to a micropipettor, scrapecells from plate and homogenize cell material by repeatedly aspiratingand dispensing the HNTG^(*) lysis buffer. Transfer lysate to a coated,blocked, and washed ELISA plate. Incubate shaking at room temperaturefor one hour.

[0346] 7. Remove lysate and wash 4 times with TBST. Transfer freshlydiluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubateshaking at room temperature for 30 minutes in the presence of theanti-Ptyr antiserum (1:3000 dilution in TBST).

[0347] 8. Remove the anti-Ptyr antibody and wash 4 times with TBST.Transfer the freshly diluted TAGO anti-rabbit IgG antibody to the ELISAplate at 100 μl per well. Incubate shaking at room temperature for 30minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST).

[0348] 9. Remove TAGO detection antibody and wash 4 times with TBST.Transfer freshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl perwell. Incubate shaking at room temperature for 20 minutes. (ABTS/H₂O₂solution: 1.0 μl 30% H₂O₂ in 10 ml ABTS stock).

[0349] 10. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), anddetermine O.D. at 410 nm.

[0350] 11. The maximal phosphotyrosine signal is determined bysubtracting the value of the negative controls from the positivecontrols. The percent inhibition of phosphotyrosine content forextract-containing wells is then calculated, after subtraction of thenegative controls.

[0351] C. PDGF-R ELISA

[0352] All cell culture media, glutamine, and fetal bovine serum can bepurchased from Gibco Life Technologies (Grand Island, N.Y.) unlessotherwise specified. All cells are grown in a humid atmosphere of 90-95%air and 5-10% CO₂ at 37° C. All cell lines are routinely subculturedtwice a week and are negative for mycoplasma as determined by theMycotect method (Gibco).

[0353] For ELISA assays, cells (U1242, obtained from JosephSchlessinger, NYU) are grown to 80-90% confluency in growth medium (MEMwith 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN) and seeded in 96-welltissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well.After overnight incubation in 0.5% serum-containing medium, cells arechanged to serum-free medium and treated with test compound for 2 hr ina 5% CO₂, 37° C. incubator. Cells are then stimulated with ligand for5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10%glycerol, 5 mM EDTA, 5 mM Na₃VO₄, 0.2% Triton X-100, and 2 mM NaPyr).Cell lysates (0.5 mg/well in PBS) are transferred to ELISA platespreviously coated with receptor-specific antibody and which had beenblocked with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and0.1% Triton X-100) at room temperature for 30 min. Lysates are incubatedwith shaking for 1 hour at room temperature. The plates are washed withTBST four times and then incubated with polyclonal anti-phosphotyrosineantibody at room temperature for 30 minutes. Excess anti-phosphotyrosineantibody is removed by rinsing the plate with TBST four times. Goatanti-rabbit IgG antibody is added to the ELISA plate for 30 min at roomtemperature followed by rinsing with TBST four more times. ABTS (100 mMcitric acid, 250 mM Na₂HPO₄ and 0.5 mg/mL2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H₂O₂ (1.2 mL30% H₂O₂ to 10 ml ABTS) is added to the ELISA plates to start colordevelopment. Absorbance at 410 nm with a reference wavelength of 630 nmis recorded about 15 to 30 min after ABTS addition.

[0354] d. IGF-I RECEPTOR ELISA

[0355] The following protocol may be used to measure phosphotyrosinelevel on IGF-I receptor, which indicates IGF-I receptor tyrosine kinaseactivity.

[0356] Materials And Reagents. The following materials and reagents areused:

[0357] a. The cell line used in this assay is 3T3/IGF-1R, a cell linegenetically engineered to overexpresses IGF-1 receptor.

[0358] b. NIH3T3/IGF-1R is grown in an incubator with 5% CO₂ at 37° C.The growth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.

[0359] c. Affinity purified anti-IGF-1R antibody 17-69.

[0360] d. D-PBS: KH₂PO₄ 0.20 g/l K₂HPO₄ 2.16 g/l KCl 0.20 g/l NaCl 8.00g/l (pH 7.2)

[0361] e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-FatDry Milk).

[0362] f. TBST buffer: Tris-HCl 50 mM NaCl 150 mM (pH 7.2/HCl 10 N)Triton X-100 0.1%

[0363]  Stock solution of TBS (10×) is prepared, and Triton X-100 isadded to the buffer during dilution.

[0364] g. HNTG buffer: HEPES 20 mM NaCl 150 mM (pH 7.2/HCl 1 N) Glycerol10% Triton X-100 0.2%

[0365]  Stock solution (5×) is prepared and kept at 4° C.

[0366] h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) as 100×stock.

[0367] I. Na₃VO₄: 0.5 M as 100×stock and aliquots are kept in −80° C.

[0368] j. Na₄P₂O₇: 0.2 M as 100×stock.

[0369] k. Insulin-like growth factor-1 from Promega (Cat# G5111).

[0370] l. Rabbit polyclonal anti-phosphotyrosine antiserum.

[0371] m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago(Cat. No. 4520, Lot No. 1802): Tago, Inc., Burlingame, Calif.

[0372] n. ABTS (2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid))solution: Citric acid 100 mM Na₂HPO₄ 250 mM (pH 4.0/1 N HCl) ABTS 0.5mg/ml

[0373]  ABTS solution should be kept in dark and 4° C. The solutionshould be discarded when it turns green.

[0374] o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4°C.

[0375] Procedure. All the following steps are conducted at roomtemperature unless it is specifically indicated. All ELISA platewashings are performed by rinsing the plate with tap water three times,followed by one TBST rinse. Pat plate dry with paper towels.

[0376] A. Cell Seeding:

[0377] 1. The cells, grown in tissue culture dish (Corning 25020-100) to80-90% confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100,GIBCO).

[0378] 2. Resuspend the cells in fresh DMEM+10% FBS+2 mM L-Glutamine,and transfer to 96-well tissue culture plate (Corning, 25806-96) at20,000 cells/well (100 μl/well). Incubate for 1 day then replace mediumto serum-free medium (90/μl) and incubate in 5% CO₂ and 37° C.overnight.

[0379] B. ELISA Plate Coating and Blocking:

[0380] 1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1RAntibody at 0.5 μg/well in 100 μl PBS at least 2 hours.

[0381] 2. Remove the coating solution, and replace with 100 μl BlockingBuffer, and shake for 30 minutes. Remove the blocking buffer and washthe plate just before adding lysate.

[0382] C. Assay Procedures:

[0383] 1. The drugs are tested in serum-free condition.

[0384] 2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-wellpoly-propylene plate, and transfer 10 μl/well of this solution to thecells to achieve final drug dilution 1:100, and final DMSO concentrationof 1.0%. Incubate the cells in 5% CO₂ at 37° C. for 2 hours.

[0385] 3. Prepare fresh cell lysis buffer (HNTG*) HNTG 2 ml EDTA 0.1 mlNa₃VO₄ 0.1 ml Na₄(P₂O₇) 0.1 ml H₂O 7.3 ml

[0386] 4. After drug incubation for two hours, transfer 10 μl/well of200 nM IGF-1 Ligand in PBS to the cells (Final Conc. =20 nM), andincubate at 5% CO₂ at 37° C. for 10 minutes.

[0387] 5. Remove media and add 100 μl/well HNTG* and shake for 10minutes. Look at cells under microscope to see if they are adequatelylysed.

[0388] 6. Use a 12-channel pipette to scrape the cells from the plate,and homogenize the lysate by repeated aspiration and dispensing.Transfer all the lysate to the antibody coated ELISA plate, and shakefor 1 hour.

[0389] 7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000with TEST) 100 μl/well, and shake for 30 minutes.

[0390] 8. Remove anti-pTyr, wash the plate, transfer TAGO (1:3,000 withTBST) 100 μl/well, and shake for 30 minutes.

[0391] 9. Remove detection antibody, wash the plate, and transfer freshABTS/H₂O₂ (1.2 μl H₂O₂ to 10 ml ABTS) 100 μl/well to the plate to startcolor development.

[0392] 10. Measure OD at 410 nm with a reference wavelength of 630 nm inDynatec MR5000.

[0393] e. EGF Receptor ELISA

[0394] EGF Receptor kinase activity in cells genetically engineered toexpress human EGF-R can be measured as described below:

[0395] Materials and Reagents. The following materials and reagents areused

[0396] a. EGF Ligand: stock concentration =16.5 μM; EGF 201, TOYOBO,Co., Ltd. Japan.

[0397] b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFRextracellular domain).

[0398] c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).

[0399] d. Detection antibody: Goat anti-rabbit lgG horse radishperoxidase conjugate, TAGO, Inc., Burlingame, Calif.

[0400] e. TBST buffer: Tris-HCl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1f. HNTG 5X stock: HEPES 0.1 M NaCl 0.75 M Glycerol 50 Triton X-100 1.0%g. ABTS stock: Citric Acid 100 mM Na₂HPO₄ 250 mM HCl, conc. 4.0 pH ABTS0.5 mg/ml

[0401] Keep solution in dark at 4° C. until used.

[0402] h. Stock reagents of:

[0403] EDTA 100 mM pH 7.0

[0404] Na₃VO₄ 0.5 M

[0405] Na₄(P₂O₇) 0.2 M

[0406] Procedure. The following protocol is used:

[0407] A. Pre-coat ELISA Plate

[0408] 1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with05-101 antibody at 0.5 μg per well in PBS, 150 μl final volume/well, andstore overnight at 4° C. Coated plates are good for up to 10 days whenstored at 4° C.

[0409] 2. On day of use, remove coating buffer and replace with blockingbuffer (5% Carnation Instant NonFat Dry Milk in PBS). Incubate theplate, shaking, at room temperature (about 23° C. to 25° C.) for 30minutes. Just prior to use, remove blocking buffer and wash plate 4times with TBST buffer.

[0410] B. Seeding Cells

[0411] 1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987)can be use for this assay.

[0412] 2. Choose dishes having 80-90% confluence for the experiment.Trypsinize cells and stop reaction by adding 10% CS DMEM medium. Suspendcells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1000rpm at room temperature for 5 minutes.

[0413] 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum),and count the cells using trypan blue. Viability above 90% isacceptable. Seed cells in DMEM medium (0.5% bovine serum) at a densityof 10,000 cells per well, 100 μl per well, in a 96 well microtiterplate. Incubate seeded cells in 5% CO₂ at 37° C. for about 40 hours.

[0414] C. Assay Procedures.

[0415] 1. Check seeded cells for contamination using an invertedmicroscope. Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium,then transfer 5 μl to a test well for a final drug dilution of 1:200 anda final DMSO concentration of 1%. Control wells receive DMSO alone.Incubate in 5% CO₂ at 37° C. for one hour.

[0416] 2. Prepare EGF ligand: dilute stock EGF in DMEM so that upontransfer of 10 μl dilute EGF (1:12 dilution), 25 nM final concentrationis attained.

[0417] 3. Prepare fresh 10 ml HNTG^(*) sufficient for 100 μl per wellwherein HNTG* comprises: HNTG stock (2.0 ml), milli-Q H₂O (7.3 ml),EDTA, 100 mM, pH 7.0 (0.5 ml), Na₃VO₄0.5 M (0.1 ml) and Na₄(P₂O₇), 0.2 M(0.1 ml).

[0418] 4. Place on ice.

[0419] 5. After two hours incubation with drug, add prepared EGF ligandto cells, 10 μl per well, to yield a final concentration of 25 nM.Control wells receive DMEM alone. Incubate, shaking, at roomtemperature, for 5 minutes.

[0420] 6. Remove drug, EGF, and DMEM. Wash cells twice with PBS.Transfer HNTG^(*) to cells, 100 μl per well. Place on ice for 5 minutes.Meanwhile, remove blocking buffer from other ELISA plate and wash withTBST as described above.

[0421] 7. With a pipette tip securely fitted to a micropipettor, scrapecells from plate and homogenize cell material by repeatedly aspiratingand dispensing the HNTG^(*) lysis buffer. Transfer lysate to a coated,blocked, and washed ELISA plate. Incubate shaking at room temperaturefor one hour.

[0422] 8. Remove lysate and wash 4 times with TBST. Transfer freshlydiluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubateshaking at room temperature for 30 minutes in the presence of theanti-Ptyr antiserum (1:3000 dilution in TBST).

[0423] 9. Remove the anti-Ptyr antibody and wash 4 times with TBST.Transfer the freshly diluted TAGO 30 anti-rabbit IgG antibody to theELISA plate at 100 μl per well. Incubate shaking at room temperature for30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST).

[0424] 10. Remove detection antibody and wash 4 times with TBST.Transfer freshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl perwell. Incubate at room temperature for 20 minutes. ABTS/H₂O₂solution:1.2 μl 30% H₂O₂in 10 ml ABTS stock.

[0425] 11. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), anddetermine O.D. at 410 nm.

[0426] 12. The maximal phosphotyrosine signal is determined bysubtracting the value of the negative controls from the positivecontrols. The percent inhibition of phosphotyrosine content forextract-containing wells is then calculated, after subtraction of thenegative controls.

[0427] f. Met Autophosphorylation Assay—ELISA

[0428] This assay determines Met tyrosine kinase activity by analyzingMet protein tyrosine kinase levels on the Met receptor.

[0429] 1. Reagents

[0430] a. HNTG (5×stock solution): Dissolve 23.83 g HEPES and 43.83 gNaCl in about 350 ml dH2O. Adjust pH to 7.2 with HCl or NaOH, add 500 mlglycerol and 10 ml Triton X-100, mix, add dH2O to 1 L total volume. Tomake 1 L of 1×working solution add 200 ml 5×stock solution to 800 mldH2O, check and adjust pH as necessary, store at 4° C.

[0431] b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco Cat. #450-1300 EB (1×solution).

[0432] c. Blocking Buffer: in 500 ml dH2O place 100 g BSA, 12.1 gTris-pH7.5, 58.44 g NaCl and 10 ml Tween-20, dilute to 1 L total volume.

[0433] d. Kinase Buffer: To 500 ml dH2O add 12.1 g TRIS pH7.2, 58.4 gNaCl, 40.7 g MgCl₂ and 1.9 g EGTA; bring to 1 L total volume with dH2O.

[0434] e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. # P-7626, to435.5 mg, add 100% ethanol to 25 ml total volume, vortex.

[0435] f. ATP (Bacterial Source), Sigma Cat. # A-7699, store powder at−20° C.; to make up solution for use, dissolve 3.31 mg in 1 ml dH₂O.

[0436] g. RC-20H HRPO Conjugated Anti-Phosphotyrosine, TransductionLaboratories Cat. # E120H.

[0437] h. Pierce 1-Step (TM) Turbo TMB-ELISA(3,3′,5,5′-tetramethylbenzidine, Pierce Cat. # 34022.

[0438] i. H₂SO₄, add 1 ml conc. (18N) to 35 ml dH2O.

[0439] j. TRIS HCL, Fischer Cat. # BP152-5; to 121.14 g of material, add600 ml MilliQ H₂O, adjust pH to 7.5 (or 7.2) with HCl, bring volume to 1L with MilliQ H₂O.

[0440] k. NaCl, Fischer Cat. # S271-10, make up 5M solution.

[0441] l. Tween-20, Fischer Cat. # S337-500.

[0442] m. Na₃VO₄, Fischer Cat. # S454-50, to 1.8 g material add 80 mlMilliQ H₂O, adjust pH to 10.0 with HCl or NaOH, boil in microwave, cool,check pH, repeat procedure until pH stable at 10.0, add MilliQ H₂O to100 ml total volume, make 1 ml aliquots and store at −80° C.

[0443] n. MgCl₂, Fischer Cat. # M33-500, make up 1M solution.

[0444] o. HEPES, Fischer Cat. # BP310-500, to 200 ml MilliQ H₂O, add59.6 g material, adjust pH to 7.5, bring volume to 250 ml total, sterilefilter.

[0445] p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30 gramsmaterial add sterile distilled water to make total volume of 300 ml,store at 40° C.

[0446] q. TBST Buffer: to approx. 900 ml dH₂O in a 1 L graduatedcylinder add 6.057 g TRIS and 8.766 g NaCl, when dissolved, adjust pH to7.2 with HCl, add 1.0 ml Triton X-100 and bring to 1 L total volume withdH₂O.

[0447] r. Goat Affinity purified antibody Rabbit IgG (whole molecule),Cappel Cat. # 55641.

[0448] s. Anti h-Met (C-28) rabbit polyclonal IgG antibody, Santa CruzChemical Cat. # SC-161.

[0449] t. Transiently Transfected EGFR/Met chimeric cells (EMR) (Komada,et al., Oncogene, 8:2381-2390 (1993).

[0450] u. Sodium Carbonate Buffer, (Na₂CO₄, Fischer Cat. # S495):

[0451] to 10.6 g material add 800 ml MilliQ H₂O, when dissolved adjustpH to 9.6 with NaOH, bring up to 1 L total volume with MilliQ H₂O,filter, store at 4° C.

[0452] 2. Procedure

[0453] All of the following steps are conducted at room temperatureunless it is specifically indicated otherwise. All ELISA plate washingis by rinsing 4× with TBST.

[0454] A. EMR Lysis

[0455] This procedure can be performed the night before or immediatelyprior to the start of receptor capture.

[0456] 1. Quick thaw lysates in a 37° C. waterbath with a swirlingmotion until the last crystals disappear.

[0457] 2. Lyse cell pellet with 1×HNTG containing 1 mM PMSF. Use 3 ml ofHNTG per 15 cm dish of cells. Add ½ the calculated HNTG volume, vortexthe tube for 1 min., add the remaining amount of HNTG, vortex foranother min.

[0458] 3. Balance tubes, centrifuge at 10,000×g for 10 min at 4° C.

[0459] 4. Pool supernatants, remove an aliquot for proteindetermination.

[0460] 5. Quick freeze pooled sample in dry ice/ethanol bath. This stepis performed regardless of whether lysate will be stored overnight orused immediately following protein determination.

[0461] 6. Perform protein determination using standard bicinchoninicacid (BCA) method (BCA Assay Reagent Kit from Pierce Chemical Cat. #23225).

[0462] B. ELISA Procedure

[0463] 1. Coat Corning 96 well ELISA plates with 5 μg per well Goatanti-Rabbit antibody in Carbonate Buffer for a total well volume of 50μl. Store overnight at 4° C.

[0464] 2. Remove unbound Goat anti-rabbit antibody by inverting plate toremove liquid.

[0465] 3. Add 150 μl of Blocking Buffer to each well. Incubate for 30min. at room temperature with shaking.

[0466] 4. Wash 4×with TBST. Pat plate on a paper towel to remove excessliquid and bubbles.

[0467] 5. Add 1 μg per well of Rabbit anti-Met antibody diluted in TBSTfor a total well volume of 100 μl.

[0468] 6. Dilute lysate in HNTG (90 μg lysate/100 μl)

[0469] 7. Add 100 μl of diluted lysate to each well. Shake at roomtemperature for 60 min.

[0470] 8. Wash 4×with TBST. Pat on paper towel to remove excess liquidand bubbles.

[0471] 9. Add 50 μl of 1×lysate buffer per well.

[0472] 10. Dilute compounds/extracts 1:10 in 1×Kinase Buffer in apolypropylene 96 well plate.

[0473] 11. Transfer 5.5 μl of diluted drug to ELISA plate wells.Incubate at room temperature with shaking for 20 min.

[0474] 12. Add 5.5 μl of 60 μM ATP solution per well. Negative controlsdo not receive any ATP. Incubate at room temperature for 90 min., withshaking.

[0475] 13. Wash 4× with TBST. Pat plate on paper towel to remove excessliquid and bubbles.

[0476] 14. Add 100 μl per well of RC20 (1:3000 dilution in BlockingBuffer). Incubate 30 min. at room temperature with shaking.

[0477] 15. Wash 4× with TBST. Pat plate on paper towel to remove excessliquid and bubbles.

[0478] 16. Add 100 μl per well of Turbo-TMB. Incubate with shaking for30-60 min.

[0479] 17. Add 100 μl per well of 1M H₂SO₄ to stop reaction.

[0480] 18. Read assay on Dynatech MR7000 ELISA reader.

[0481] Test Filter=450 nm, reference filter=410 nm.

[0482] g. Biochemical arc assay—ELISA

[0483] This assay is used to determine src protein kinase activitymeasuring phosphorylation of a biotinylated peptide as the readout.

[0484] Materials and Reagents:

[0485] a. Yeast transformed with src from Courtneidge Laboratory (Sugen,Inc., Redwood City, Calif.).

[0486] b. Cell lysates: Yeast cells expressing src are pelleted, washedonce with water, re-pelleted and stored at −80° C. until use.

[0487] c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared bystandard procedures well known to those skilled in the art.

[0488] d. DMSO: Sigma, St. Louis, Mo.

[0489] e. 96 Well ELISA Plate: Corning 96 Well Easy Wash, Modified flatBottom Plate, Corning Cat. #25805-96.

[0490] f. NUNC 96-well V-bottom polypropylene plates for dilution ofcompounds: Applied Scientific Cat. # A-72092.

[0491] g. Vecastain ELITE ABC reagent: Vector, Burlingame, Calif.

[0492] h. Anti-src (327) mab: Schizosaccharomyces Pombe is used toexpress recombinant Src (Superti-Furga, et al., EMBO J., 12:2625-2634;Superti-Furga, et al., Nature Biochem., 14:600-605). S. Pombe strainSP200 (h-s leul.32 ura4 ade210) is grown as described andtransformations are pRSP expression plasmids are done by the lithiumacetate method (Superti-Furga, supra). Cells are grown in the presenceof 1 μM thiamine to repress expression from the nmtl promoter or in theabsence of thiamine to induce expression.

[0493] i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be usedinstead).

[0494] j. Turbo TMB-ELISA peroxidase substrate: Pierce Chemical.

[0495] 2. Buffer Solutions:

[0496] a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCOCat. # 450-1300EB.

[0497] b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS.

[0498] c. Carbonate Buffer: Na₂CO₄ from Fischer, Cat. # S495, make up100 mM stock solution.

[0499] d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl₂; 0.2 ml(from a 1M stock solution) MnCl₂; 0.2 ml (from a 1M stock solution) DTT;5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 mltotal volume with MilliQ H₂O.

[0500] e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 mlNaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 mlEDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stocksolution); 0.1 ml Na₃VO₄ (from a 0.1 M stock solution); bring to 100 mltotal volume with MilliQ H₂O.

[0501] f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51mg/ml)

[0502] g. TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H₂O add121.14 g material, adjust pH to 7.5 with HCl, bring to 1 L total volumewith MilliQ H₂O.

[0503] h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution withMilliQ H₂O.

[0504] i. Na₃VO₄: Fischer Cat. # S454-50; to 80 ml MilliQ H₂O, add 1.8 gmaterial; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool;check pH, repeat pH adjustment until pH remains stable afterheating/cooling cycle; bring to 100 ml total volume with MilliQ H₂O;make 1 ml aliquots and store at −80° C.

[0505] j. MgCl₂: Fischer Cat. # M33-500, make up 1M stock solution withMilliQ H₂O.

[0506] k. HEPES: Fischer Cat. # BP 310-500; too 200 ml MilliQ H2O, add59.6 g material, adjust pH to 7.5, bring to 250 ml total volume withMilliQ H₂O, sterile filter (1M stock solution).

[0507] l. TBST Buffer: TBST Buffer: To 900 ml dH₂O add 6.057 g TRIS and8.766 g NaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bringto 1 L total volume with dH₂O.

[0508] m. MnCl₂: Fischer Cat. # M87-100, make up 1M stock solution withMilliQ H₂O.

[0509] n. DTT: Fischer Cat. # BP172-5.

[0510] o. TBS (TRIS Buffered Saline): to 900 ml MilliQ H₂O add 6.057 gTRIS and 8.777 g NaCl; bring to 1 L total volume with MilliQ H₂O.

[0511] p. Kinase Reaction Mixture: Amount per assay plate (100 wells):1.0 ml Kinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml withMilliQ H₂O.

[0512] q. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide stocksolution (1 mM, 2.98 mg/ml) in water fresh just before use.

[0513] r. Vectastain ELITE ABC reagent: To prepare 14 ml of workingreagent, add 1 drop of reagent A to 15 ml TBST and invert tube severaltimes to mix. Then add 1 drop of reagent B. Put tube on orbital shakerat room temperature and mix for 30 minutes.

[0514] 3. Procedures:

[0515] a. Preparation of src coated ELISA plate.

[0516] 1. Coat ELISA plate with 0.5μg/well anti-src mab in 100 μl of pH9.6 sodium carbonate buffer at 4° C. overnight.

[0517] 2. Wash wells once with PBS.

[0518] 3. Block plate with 0.15 ml 5% milk in PBS for 30 min. at roomtemperature.

[0519] 4. Wash plate 5× with PBS.

[0520] 5. Add 10 μg/well of src transformed yeast lysates diluted inLysis Buffer (0.1 ml total volume per well). (Amount of lysate may varybetween batches.) Shake plate for 20 minutes at room temperature.

[0521] b. Preparation of phosphotyrosine antibody-coated ELISA plate.

[0522] 1. 4G10 plate: coat 0.5 μg/well 4G10 in 100 μl PBS overnight at4° C. and block with 150 μl of 5% milk in PBS for 30 minutes at roomtemperature.

[0523] C. Kinase assay procedure.

[0524] 1. Remove unbound proteins from step 1-7, above, and wash plates5×with PBS.

[0525] 2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μlof 10×Kinase Buffer and 10 μM (final concentration)biotin-EEEYEEYEEEYEEEYEEEY per well diluted in water.

[0526] 3. Add 10 μl of compound diluted in water containing 10% DMSO andpre-incubate for 15 minutes at room temperature.

[0527] 4. Start kinase reaction by adding 10 μl/well of 0.05 mM ATP inwater (5 μM ATP final).

[0528] 5. Shake ELISA plate for 15 min. at room temperature.

[0529] 6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well.

[0530] 7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISAplate from section B, above.

[0531] 8. Incubate for 30 min. while shaking at room temperature.

[0532] 9. Wash plate 5× with TBST.

[0533] 10. Incubate with Vectastain ELITE ABC reagent (100 μl/well) for30 min. at room temperature.

[0534] 11. Wash the wells 5× with TBST.

[0535] 12. Develop with Turbo TMB.

[0536] h. Biochemical lck Assay—ELISA

[0537] This assay is used to determine lck protein kinase activitiesmeasuring phosphorylation of GST-ζ as the readout.

[0538] 1. Materials and Reagents:

[0539] a. Yeast transformed with lck. Schizosaccharomyces Pombe is usedto express recombinant Lck (Superti-Furga, et al., EMBO J, 12:2625-2634;Superti-Furga, et al., Nature Biotech., 14:600-605). S. Pombe strainSP200 (h-s leul.32 ura4 ade210) is grown as described andtransformations with pRSP expression plasmids are done by the lithiumacetate method (Superti-Furga, supra). Cells are grown in the presenceof 1 μM thiamine to induce expression.

[0540] b. Cell lysates: Yeast cells expressing lck are pelleted, washedonce in water, re-pelleted and stored frozen at −80° C. until use.

[0541] c. GST-ζ: DNA encoding for GST-ζ fusion protein for expression inbacteria obtained from Arthur Weiss of the Howard Hughes MedicalInstitute at the University of California, San Francisco. Transformedbacteria are grown overnight while shaking at 25° C. GST-ζ is purifiedby glutathione affinity chromatography, Pharmacia, Alameda, Calif.

[0542] d. DMSO: Sigma, St. Louis, Mo.

[0543] e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified FlatBottom Plate, Corning Cat. #25805-96.

[0544] f. NUNC 96-well V-bottom polypropylene plates for dilution ofcompounds: Applied Scientific Cat. # AS-72092.

[0545] g. Purified Rabbit anti-GST antiserum: Amrad Corporation(Australia) Cat. #90001605.

[0546] h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301.

[0547] i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. # 5215-005-003.

[0548] j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat # sc-433.

[0549] k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40 may be usedinstead).

[0550] 2. Buffer solutions:

[0551] a. PBS (Dulbecco's Phosphate-Buffered Saline) 1X solution: GIBCOPBS, GIBCO Cat. # 450-1300EB.

[0552] b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS-pH7.5, 58.44 g NaCl,10 ml Tween-20, bring up to 1 L total volume with MilliQ H₂O.

[0553] c. Carbonate Buffer: Na₂CO₄ from Fischer, Cat. # S495;

[0554] make up 100 mM solution with MilliQ H₂O.

[0555] d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl₂; 0.2 ml(from a 1M stock solution) MnCl₂; 0.2 ml (from a 1M stock solution) DTT;5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 mltotal volume with MilliQ H₂O.

[0556] e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 mlNaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 mlEDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stocksolution); 0.1 ml Na₃VO₄ (from a 0.1 M stock solution); bring to 100 mltotal volume with MilliQ H₂O.

[0557] f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51mg/ml).

[0558] g. TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H₂O add121.14 g material, adjust pH to 7.5 with HCl, bring to 1 L total volumewith MilliQ H₂O.

[0559] h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution withMilliQ H₂O.

[0560] i. Na₃VO₄: Fischer Cat. # S454-50; to 80 ml MilliQ H₂O, add 1.8 gmaterial; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool;check pH, repeat pH adjustment until pH remains stable afterheating/cooling cycle; bring to 100 ml total volume with MilliQ H₂O;make 1 ml aliquots and store at −80° C.

[0561] j. MgCl₂: Fischer Cat. # M33-500, make up 1M stock solution withMilliQ H₂O.

[0562] k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ H₂O, add59.6 g material, adjust pH to 7.5, bring to 250 ml total volume withMilliQ H₂O, sterile filter (1M stock solution).

[0563] l. Albumin, Bovine (BSA), Sigma Cat. # A4503; to 150 ml MilliQH₂O add 30 g material, bring 300 ml total volume with MilliQ H₂O, filterthrough 0.22 μm filter, store at 4° C.

[0564] m. TBST Buffer: To 900 ml dH₂O add 6.057 g TRIS and 8.766 g NaCl;adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L totalvolume with dH₂O.

[0565] n. MnCl₂: Fischer Cat. # M87-100, make up 1M stock solution withMilliQ H₂O.

[0566] o. DTT; Fischer Cat. # BP172-5.

[0567] p. TBS (TRIS Buffered Saline): to 900 ml MilliQ H₂O add 6.057 gTRIS and 8.777 g NaCl; bring to 1 L total volume with MilliQ H₂O.

[0568] q. Kinase Reaction Mixture: Amount per assay plate (100 wells):1.0 ml Kinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml withMilliQ H₂O.

[0569]2. Procedures:

[0570] a. Preparation of Lck coated ELISA plate.

[0571] 1. Coat 2.0 μg/well Sheep anti-mouse IgG in 100 μl of pH 9.6sodium carbonate buffer at 4° C. overnight.

[0572] 2. Wash well once with PBS.

[0573] 3. Block plate with 0.15 ml of blocking Buffer for 30 min. atroom temp.

[0574] 4. Wash plate 5×with PBS.

[0575] 5. Add 0.5 μg/well of anti-lck (mab 3A5) in 0.1 ml PBS at roomtemperature for 1-2 hours.

[0576] 6. Wash plate 5×with PBS.

[0577] 7. Add 20 μg/well of lck transformed yeast lysates diluted inLysis Buffer (0.1 ml total volume per well). (Amount of lysate may varybetween batches) Shake plate at 4° C. overnight to prevent loss ofactivity.

[0578] b. Preparation of phosphotyrosine antibody-coated ELISA plate.

[0579] 1. UB40 plate: 1.0 μg/well UB40 in 100 μl of PBS overnight at 4°C. and block with 150 μl of Blocking Buffer for at least 1 hour.

[0580] c. Kinase assay procedure.

[0581] 1. Remove unbound proteins from step 1-7, above, and wash plates5× with PBS.

[0582] 2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μlof 10×Kinase Buffer and 2 μg GST-ζ per well diluted with water).

[0583] 3. Add 10 μl of compound diluted in water containing 10% DMSO andpre-incubate for 15 minutes at room temperature.

[0584] 4. Start kinase reaction by adding 10 μl/well of 0.1 mM ATP inwater (10 μM ATP final).

[0585] 5. Shake ELISA plate for 60 min. at room temperature.

[0586] 6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well.

[0587] 7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISAplate from section B, above.

[0588] 8. Incubate while shaking for 30 min. at room temperature.

[0589] 9. Wash plate 5× with TBST.

[0590] 10. Incubate with Rabbit anti-GST antibody at 1:5000 dilution in100 μl TBST for 30 min. at room temperature.

[0591] 11. Wash the wells SX with TBST.

[0592] 12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilutionin 100 μl of TBST for 30 min. at room temperature.

[0593] 13. Wash the wells 5× with TBST.

[0594] 14. Develop with Turbo TMB.

[0595] i. ASSAY MEASURING PHOSPHORYLATING FUNCTION OF RAF

[0596] The following assay reports the amount of RAF-catalyzedphosphorylation of its target protein MEK as well as MEK's target MAPK.The RAF gene sequence is described in Bonner et al., 1985, Molec. Cell.Biol. 5: 1400-1407, and is readily accessible in multiple gene sequencedata banks. Construction of the nucleic acid vector and cell linesutilized for this portion of the invention are fully described inMorrison et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8855-8859.

Materials and Reagents

[0597] 1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg,Md.

[0598] 2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol,1 mM PMSF, 5 mg/L Aprotenin, 0.5% Triton X-100;

[0599] 3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression andpurification by affinity chromatography are performed according to themanufacturer's procedures. Catalog# K 350-01 and R 350-40, InvitrogenCorp., San Diego, Calif.

[0600] 4. His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Bluecells transformed with pUC18 vector encoding His-MAPK. His-MAPK ispurified by Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia,Alameda, Calif., as described herein.

[0601] 5. Sheep anti mouse IgG: Jackson laboratories, West Grove, Pa.Catalog, # 515-006-008, Lot# 28563

[0602] 6. RAF-1 protein kinase specific antibody: URP2653 from UBI.

[0603] 7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL,Gaithersburg, Md.

[0604] 8. Wash buffer: TBST - 50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1%Triton X-100

[0605] 9. Block buffer: TBST, 0.1% ethanolamine pH 7.4

[0606] 10. DMSO, Sigma, St. Louis, Mo.

[0607] 11. Kinase buffer (KB): 20 mM HEPES/HCl pH 7.2, 150 mM NaCl, 0.1%Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium ortho vanadate,0.5 MM DTT and 10 mM MgCl₂.

[0608] 12. ATP mix: 100 mM MgCl₂, 300 mM ATP, 10 mCi 33P ATP(Dupont-NEN)/mL.

[0609] 13 Stop solution: 1% phosphoric acid; Fisher, Pittsburgh, Pa.

[0610] 14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku,Finnland.

[0611] 15. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh,Pa.

[0612] 16. Tomtec plate harvester, Wallac, Turku, Finnland.

[0613] 17. Wallac beta plate reader # 1205, Wallac, Turku, Finnland.

[0614] 18. NUNC 96-well V bottom polypropylene plates for compoundsApplied Scientific Catalog # AS-72092.

Procedure

[0615] All of the following steps are conducted at room temperatureunless specifically indicated.

[0616] 1. ELISA plate coating: ELISA wells are coated with 100 ml ofSheep anti mouse affinity purified antiserum (1 mg/100 mL coatingbuffer) over night at 4° C. ELISA plates can be used for two weeks whenstored at 4° C.

[0617] 2. Invert the plate and remove liquid. Add 100 mL of blockingsolution and incubate for 30 min.

[0618] 3. Remove blocking solution and wash four times with wash buffer.Pat the plate on a paper towel to remove excess liquid.

[0619] 4. Add 1 mg of antibody specific for RAF-1 to each well andincubate for 1 hour. Wash as described in step 3.

[0620] 5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute withTBST to 10 mg/100 mL. Add 10 mg of diluted lysate to the wells andincubate for 1 hour. Shake the plate during incubation. Negativecontrols receive no lysate. Lysates from RAS/RAF infected Sf9 insectcells are prepared after cells are infected with recombinantbaculoviruses at a MOI of 5 for each virus, and harvested 48 hourslater. The cells are washed once with PBS and lysed in RIPA buffer.Insoluble material is removed by centrifugation.(5 min at 10 000×g).Aliquots of lysates are frozen in dry ice/ethanol and stored at −80° C.until use.

[0621] 6. Remove non-bound material and wash as outlined above (step 3).

[0622] 7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjustthe volume to 40 mL with kinase buffer. Methods for purifying T-MEK andMAPK from cell extracts are provided herein by example.

[0623] 8. Pre-dilute compounds (stock solution 10 mg/mL DMSO) orextracts 20 fold in TBST plus 1% DMSO. Add 5 mL of the pre-dilutedcompounds/extracts to the wells described in step 6. Incubate for 20min. Controls receive no drug.

[0624] 9. Start the kinase reaction by addition of 5 mL ATP mix; Shakethe plates on an ELISA plate shaker during incubation.

[0625] 10. Stop the kinase reaction after 60 min by addition of 30 mLstop solution to each well.

[0626] 11. Place the phosphocellulose mat and the ELISA plate in theTomtec plate harvester. Harvest and wash the filter with the filter washsolution according to the manufacturers recommendation. Dry the filtermats. Seal the filter mats and place them in the holder. Insert theholder into radioactive detection apparatus and quantify the radioactivephosphorous on the filter mats.

[0627] Alternatively, 40 mL aliquots from individual wells of the assayplate can be transferred to the corresponding positions on thephosphocellulose filter mat. After air drying the filters, put thefilters in a tray. Gently rock the tray, changing the wash solution at15 min intervals for 1 hour. Air-dry the filter mats. Seal the filtermats and place them in a holder suitable for measuring the radioactivephosphorous in the samples. Insert the holder into a detection deviceand quantify the radioactive phosphorous on the filter mats.

[0628] j. CDK2/Cyclin A—Inhibition Assay

[0629] This assay analyzes the protein kinase activity of CDK2 inexogenous substrate.

Reagents

[0630] A. Buffer A (80 mM Tris ( pH 7.2), 40 mM MgCl₂): 4.84 G. Tris(F.W.=121.1 g/mol), 4.07 g. MgCl₂ (F.W.=203.31 g/mol) dissolved in 500ml H₂O. Adjust pH to 7.2 with HCl.

[0631] B. Histone H1 solution (0.45 mg/ml Histone H1 and 20 mM HEPES pH7.2 (pH 7.4 is OK): 5 mg Histone H1 (Boehinger Mannheim) in 11.111 ml 20mM HEPES pH 7.2 (477 mg HEPES (F.W.=238.3 g/mol) dissolved in 100 mlddH₂O, stored in 1 ml aliquots at −80° C.

[0632] C. ATP solution (60 μM ATP, 300 μg/ml BSA, 3 mM DTT): 120 μl 10mM ATP, 600 μl 10 mg/ml BSA to 20 ml, stored in 1 ml aliquots at −80° C.

[0633] D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mMNaCl, 0.5 mM DTT, 10% glycerol, stored in 9 μl aliquots at −80° C.

Description of Assay

[0634] 1. Prepare solutions of inhibitors at three times the desiredfinal assay concentration in ddH₂O/15% DMSO by volume.

[0635] 2. Dispense 20 μl of inhibitors to wells of polypropylene 96-wellplates (or 20 μl 15% DMSO for positive and negative controls).

[0636] 3. Thaw Histone H1 solution (1 ml/plate), ATP solution (1ml/plate plus 1 aliquot for negative control), and CDK2 solution (9μl/plate). Keep CDK2 on ice until use. Aliquot CDK2 solutionappropriately to avoid repeated freeze-thaw cycles.

[0637] 4. Dilute 9 μl CDK2 solution into 2.1 ml Buffer A (per plate).Mix. Dispense 20 μl into each well.

[0638] 5. Mix 1 ml Histone H1 solution with 1 ml ATP solution (perplate) into a 10 ml screw cap tube. Add γ³³P ATP to a concentration of0.15 μCi/20 μl (0.15 μCi/well in assay). Mix carefully to avoid BSAfrothing. Add 20 μl to appropriate wells. Mix plates on plate shaker.For negative control, mix ATP solution with an equal amount of 20 mMHEPES pH 7.2 and add γ³³P ATP to a concentration of 0.15 μCi/20 μlsolution. Add 20 μl to appropriate wells.

[0639] 6. Let reactions proceed for 60 minutes.

[0640] 7. Add 35 μl 10% TCA to each well. Mix plates on plate shaker.

[0641] 8. Spot 40 μl of each sample onto P30 filter mat squares. Allowmats to dry (approx. 10-20 minutes).

[0642] 9 Wash filter mats 4×10 minutes with 250 ml 1% phosphoric acid(10 ml phosphoric acid per liter ddH₂O).

[0643] 10. Count filter mats with beta plate reader.

[0644] 2. Cellular/Biologic Assays

Assay 1: PDGF-Induced BrdU Incorporation Assay Materials and Reagents

[0645] (1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.

[0646] (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat.

[0647] No. 1 647 229, Boehringer Mannheim, Germany.

[0648] (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0649] (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated withperoxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0650] (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready touse, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0651] (6) PBS Washing Solution: 1X PBS, pH 7.4, made in house (Sugen,Inc., Redwood City, Calif.).

[0652] (7) Albumin, Bovine (BSA): fraction V powder; A-8551, SigmaChemical Co., USA.

[0653] (8) 3T3 cell line genetically engineered to express human PDGF-R.

Protocol

[0654] (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Glnin a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

[0655] (2) After 24 hours, the cells are washed with PBS, and then areserum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24hours.

[0656] (3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1%BSA) and test compounds are added to the cells simultaneously. Thenegative control wells receive serum free DMEM with 0.1% BSA only; thepositive control cells receive the ligand (PDGF) but no test compound.Test compounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

[0657] (4) After 20 hours of ligand activation, diluted BrdU labelingreagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubatedwith BrdU (final concentration=10 μM) for 1.5 hours.

[0658] (5) After incubation with labeling reagent, the medium is removedby decanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

[0659] (6) The FixDenat solution is thoroughly removed by decanting andtapping the inverted plate on a paper towel. Milk is added (5%dehydrated milk in PBS, 200 μl/well) as a blocking solution and theplate is incubated for 30 minutes at room temperature on a plate shaker.

[0660] (7) The blocking solution is removed by decanting and the wellsare washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS,1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutesat room temperature on a plate shaker.

[0661] (8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

[0662] (9) TMB substrate solution is added (100 μl/well) and incubatedfor 20 minutes at room temperature on a plate shaker until colordevelopment is sufficient for photometric detection.

[0663] (10) The absorbance of the samples are measured at 410 nm (in“dual wavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 2: EGF-Induced BrdU Incorporation Assay Materials and Reagents

[0664] (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan.

[0665] (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0666] (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0667] (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated withperoxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0668] (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready touse, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0669] (6) PBS Washing Solution: 1X PBS, pH 7.4, made in house (Sugen,Inc., Redwood City, Calif.).

[0670] (7) Albumin, Bovine (BSA): fraction V powder; A-8551, SigmaChemical Co., USA.

[0671] (8) 3T3 cell line genetically engineered to express human EGF-R.

Protocol

[0672] (1) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln inDMEM, in a 96 well plate. Cells are incubated overnight at 37° C. in 5%CO₂.

[0673] (2) After 24 hours, the cells are washed with PBS, and then areserum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24hours.

[0674] (3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA)and test compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (EGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

[0675] (4) After 20 hours of ligand activation, diluted BrdU labelingreagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubatedwith BrdU (final concentration=10 μM) for 1.5 hours.

[0676] (5) After incubation with labeling reagent, the medium is removedby decanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

[0677] (6) The FixDenat solution is thoroughly removed by decanting andtapping the inverted plate on a paper towel. Milk is added (5%dehydrated milk in PBS, 200 μl/well) as a blocking solution and theplate is incubated for 30 minutes at room temperature on a plate shaker.

[0678] (7) The blocking solution is removed by decanting and the wellsare washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS,1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutesat room temperature on a plate shaker.

[0679] (8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

[0680] (9) TMB substrate solution is added (100 μl/well) and incubatedfor 20 minutes at room temperature on a plate shaker until colordevelopment is sufficient for photometric detection.

[0681] (10) The absorbance of the samples are measured at 410 nm (in“dual wavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 3: EGF-Induced Her2-Driven BrdU Incorporation Materials andReagents

[0682] (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan

[0683] (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0684] (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0685] (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated withperoxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0686] (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready touse, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0687] (6) PBS Washing Solution: 1X PBS, pH 7.4, made in house.

[0688] (7) Albumin, Bovine (BSA): fraction V powder; A-8551, SigmaChemical Co., USA.

[0689] (8) 3T3 cell line engineered to express a chimeric receptorhaving the extra-cellular domain of EGF-R and the intra-cellular domainof Her2.

Protocol

[0690] (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Glnin a 96-well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

[0691] (2) After 24 hours, the cells are washed with PBS, and then areserum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24hours.

[0692] (3) on day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA)and test compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (EGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

[0693] (4) After 20 hours of ligand activation, diluted BrdU labelingreagent (1:100 in DMEM, 01% BSA) is added and the cells are incubatedwith BrdU (final concentration=10 μM) for 1.5 hours.

[0694] (5) After incubation with labeling reagent, the medium is removedby decanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

[0695] (6) The FixDenat solution is thoroughly removed by decanting andtapping the inverted plate on a paper towel. Milk is added (5%dehydrated milk in PBS, 200 μl/well) as a blocking solution and theplate is incubated for 30 minutes at room temperature on a plate shaker.

[0696] (7) The blocking solution is removed by decanting and the wellsare washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS,1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutesat room temperature on a plate shaker.

[0697] (8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

[0698] (9) TMB substrate solution is added (100 μl/well) and incubatedfor 20 minutes at room temperature on a plate shaker until colordevelopment is sufficient for photometric detection.

[0699] (10) The absorbance of the samples are measured at 410 nm (in“dual wavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 4: IGF1-Induced BrdU Incorporation Assay Materials and Reagents

[0700] (1) IGF1 Ligand: human, recombinant; G511, Promega Corp,. USA.

[0701] (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0702] (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647229, Boehringer Mannheim, Germany.

[0703] (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated withperoxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0704] (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready touse, Cat. No. 1 647 229, Boehringer Mannheim, Germany.

[0705] (6) PBS Washing Solution: 1X PBS, pH 7.4, made in house (Sugen,Inc., Redwood City, Calif.).

[0706] (7) Albumin, Bovine (BSA): fraction V powder; A-8551, SigmaChemical Co., USA.

[0707] (8) 3T3 cell line genetically engineered to express human IGF-1receptor.

Protocol

[0708] (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Glnin a 96- well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

[0709] (2) After 24 hours, the cells are washed with PBS, and then areserum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24hours.

[0710] (3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1%BSA) and test compounds are added to the cells simultaneously. Thenegative control wells receive serum free DMEM with 0.1% BSA only; thepositive control cells receive the ligand (IGF1) but no test compound.Test compounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

[0711] (4) After 16 hours of ligand activation, diluted BrdU labelingreagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubatedwith BrdU (final concentration=10 μM) for 1.5 hours.

[0712] (5) After incubation with labeling reagent, the medium is removedby decanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

[0713] (6) The FixDenat solution is thoroughly removed by decanting andtapping the inverted plate on a paper towel. Milk is added (5%dehydrated milk in PBS, 200 μl/well) as a blocking solution and theplate is incubated for 30 minutes at room temperature on a plate shaker.

[0714] (7) The blocking solution is removed by decanting and the wellsare washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS,1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutesat room temperature on a plate shaker.

[0715] (8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

[0716] (9) TMB substrate solution is added (100 μl/well) and incubatedfor 20 minutes at room temperature on a plate shaker until colordevelopment is sufficient for photometric detection.

[0717] (10) The absorbance of the samples are measured at 410 nm (in“dual wavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

[0718] g. HUV-EC-C Assay

[0719] The following protocol may also be used to measure a compound'sactivity against PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of whichare naturally expressed by HUV-EC cells.

DAY 0

[0720] 1. Wash and trypsinize HUV-EC-C cells (human umbilical veinendothelial cells, (American Type Culture Collection; catalogue no. 1730CRL). Wash with Dulbecco's phosphate-buffered saline (D-PBS; obtainedfrom Gibco BRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm² oftissue culture flask. Trypsinize with 0.05% try sin-EDTA innon-enzymatic cell dissociation solution (Sigma Chemical Company;catalogue no. C-1544). The 0.05% trypsin is made by diluting 0.25%trypsin/1 mM EDTA (Gibco; catalogue no. 25200-049) in the celldissociation solution. Trypsinize with about 1 ml/25-30 cm² of tissueculture flask for about 5 minutes at 37° C. After cells have detachedfrom the flask, add an equal volume of assay medium and transfer to a 50ml sterile centrifuge tube (Fisher Scientific; catalogue no. 05-539-6).

[0721] 2. Wash the cells with about 35 ml assay medium in the 50 mlsterile centrifuge tube by adding the assay medium, centrifuge for 10minutes at approximately 200 g, aspirate the supernatant, and resuspendwith 35 ml D-PBS. Repeat the wash two more times with D-PBS, resuspendthe cells in about 1 ml assay medium/15 cm² of tissue culture flask.Assay medium consists of F12K medium (Gibco BRL; catalogue no.21127-014)+0.5% heat-inactivated fetal bovine serum. Count the cellswith a Coulter Counter, Coulter Electronics, Inc.) and add assay mediumto the cells to obtain a concentration of 0.8-1.0×10⁵ cells/ml.

[0722] 3. Add cells to 96-well flat-bottom plates at 100 μl/well or0.8-1.0×10⁴ cells/well; incubate ˜24 h at 37° C., 5% CO₂.

DAY 1

[0723] 1. Make up two-fold drug titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of drugat 200 μM (4×the final well concentration) to the top well of aparticular plate column. Since the stock drug concentration is usually20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

[0724] Therefore, diluent made up to 2% DMSO in assay medium (F12K+0.5%fetal bovine serum) is used as diluent for the drug titrations in orderto dilute the drug but keep the DMSO concentration constant. Add thisdiluent to the remaining wells in the column at 60 μl/well. Take 60 μlfrom the 120 μl of 200 μM drug dilution in the top well of the columnand mix with the 60 μl in the second well of the column. Take 60 μl fromthis well and mix with the 60 μl in the third well of the column, and soon until two-fold titrations are completed. When the next-to-the-lastwell is mixed, take 60 μl of the 120 μl in this well and discard it.Leave the last well with 60 μl of DMSO/media diluent as anon-drug-containing control. Make 9 columns of titrated drug, enough fortriplicate wells each for 1) VEGF (obtained from Pepro Tech Inc.,catalogue no. 100-200, 2) endothelial cell growth factor (ECGF) (alsoknown as acidic fibroblast growth factor, or aFGF) (obtained fromBoehringer Mannheim Biochemica, catalogue no. 1439 600); or, 3) humanPDGF B/B (1276-956, Boehringer Mannheim, Germany) and assay mediacontrol. ECGF comes as a preparation with sodium heparin.

[0725] 2. Transfer 50 μl/well of the drug dilutions to the 96-well assayplates containing the 0.8-1.0×10⁴ cells/100 μl/well of the HUV-EC-Ccells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

[0726] 3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF,or media control to each drug condition. As with the drugs, the growthfactor concentrations are 4× the desired final concentration. Use theassay media from day 0 step 2 to make the concentrations of growthfactors. Incubate approximately 24 hours at 37° C., 5% CO₂. Each wellwill have 50 μl drug dilution, 50 μl growth factor or media, and 100 ulcells,=200 ul/well total. Thus the 4× concentrations of drugs and growthfactors become 1×once everything has been added to the wells.

DAY 2

[0727] 1. Add ³H-thymidine (Amersham; catalogue no. TRK-686) at 1μCi/well (10 μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

DAY 3

[0728] 1. Freeze plates overnight at −20° C.

DAY 4

[0729] 1. Thaw plates and harvest with a 96-well plate harvester (TomtecHarvester 96®) onto filter mats (Wallac; catalogue no. 1205-401); readcounts on a Wallac Betaplate™ liquid scintillation counter.

[0730] 3. In Vivo Animal Models

[0731] A. Xenograft Animal Models

[0732] The ability of human tumors to grow as xenografts in athymic mice(e.g., Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastrointestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

[0733] Suitable cell lines for subcutaneous xenograft experimentsinclude C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC #CRL 1619), A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6cells (lung, ATCC # HTB 56) , PC3 cells (prostate, ATCC # CRL 1435),SKOV3TP5 cells and NIH 3T3 fibroblasts genetically engineered tooverexpress EGFR, PDGFR, IGF-1R or any other test kinase. The followingprotocol can be used to perform xenograft experiments:

[0734] Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

[0735] Cell lines are grown in appropriate medium (for example, MEM,DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2mM glutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

[0736] Cells are harvested at or near confluency with 0.05% Trypsin-EDTAand pelleted at 450×g for 10 min. Pellets are resuspended in sterile PBSor media (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50-100 μL excipient (DMSO,or VPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

[0737] B. Tumor Invasion Model

[0738] The following tumor invasion model has been developed and maybeused for the evaluation of therapeutic value and efficacy of thecompounds identified to selectively inhibit KDR/FLK-1 receptor.

Procedure

[0739] 8 week old nude mice (female) (Simonsen Inc.) are used asexperimental animals. Implantation of tumor cells can be performed in alaminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kgketamine and 5 mg/kg Xylazine) are administered intraperitoneally. Amidline incision is done to expose the abdominal cavity (approximately1.5 cm in length) to inject 10⁷ tumor cells in a volume of 100 μlmedium. The cells are injected either into the duodenal lobe of thepancreas or under the serosa of the colon. The peritoneum and musclesare closed with a 6-0 silk continuous suture and the skin is closed byusing wound clips. Animals are observed daily.

Analysis

[0740] After 2-6 weeks, depending on gross observations of the animals,the mice are sacrificed, and the local tumor metastases, to variousorgans (lung, liver, brain, stomach, spleen, heart, muscle) are excisedand analyzed (measurements of tumor size, grade of invasion,immunochemistry, and in situ hybridization).

[0741] D. Measurement Of Cell Toxicity

[0742] Therapeutic compounds should be more potent in inhibitingreceptor tyrosine kinase activity than in exerting a cytotoxic effect. Ameasure of the effectiveness and cell toxicity of a compound can beobtained by determining the therapeutic index: IC₅₀/LD₅₀, IC₅₀, the doserequired to achieve 50% inhibition, can be measured using standardtechniques such as those described herein. LD₅₀the dosage which resultsin 50% toxicity, can also be measured by standard techniques (Mossman,1983, J. Immunol. Methods, 65:55-63), by measuring the amount of LDHreleased (Korzeniewski and Callewaert, 1983, J. Immunol. Methods,64:313; Decker and Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61),or by measuring the lethal dose in animal models. Compounds with a largetherapeutic index are preferred. The therapeutic index should be greaterthan 2, preferably at least 10, more preferably at least 50.

Conclusion

[0743] Thus, it will be appreciated that the compounds of the presentinvention are expected to show improved hydrosolubility and thecompounds, methods and pharmacological compositions of the presentinvention are expected to modulate PTK activity and therefore to beeffective as therapeutic agents against PTK-related disorders.

[0744] Although certain embodiments and examples have been used todescribe the present invention, it will be apparent to those skilled inthe art that changes to the embodiments and examples shown may be madewithout departing from the scope and spirit of the invention.

[0745] Other embodiments are within the following claims.

What is claimed:
 1. A 3-heteroarylidenyl-2-indolinone compound havingthe following chemical structure:

wherein, A is selected from the group consisting of nitrogen, oxygen andsulfur and it is understood that when A is oxygen or sulfur, R³ does notexist; B, D, E, F and G are independently selected from the groupconsisting of carbon and nitrogen and it is understood that when B, D,E, F or G is nitrogen, R⁴, R⁵, R⁶ or R⁷, respectively, do not exist; Zis selected from the group consisting of oxygen, sulfur and NR¹¹wherein, R¹¹ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, carbonyl, C-carboxy,O-carboxy, C-amido, guanyl, sulfonyl and trihalomethanesulfonyl; R¹ isselected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, hydroxy, alkoxy, C-carboxy, C-amido,trihalomethanecarbonyl, trihalomethane-sulfonyl and sulfonyl; R² isselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryland halogen; When A is nitrogen, R³ is selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, hydroxy, alkoxy,aryloxy, carbonyl, C-carboxy, O-carboxy, C-amido, guanyl, sulfonyl andtrihalomethanesulfonyl; One or two of R⁴, R⁵, R⁶ and R⁷ areindependently selected from the group consisting of —NR⁸R⁹,—J(CH₂)_(m)—NR⁸R⁹, —JCH₂)_(m)C(═Y)Q, —N═CNR⁸R⁹ and —NHR¹⁰; wherein, J isselected from the group consisting of oxygen, NH and sulfur; m is 0, 1,2 or 3; Y is selected from the group consisting of NH and oxygen; Q isselected from the group consisting of hydroxy, alkoxy, aryloxy, amino,N-hydroxylamino, O-carboxy, NR⁸R⁹ and N-peptidyl; R⁸ and R⁹ areindependently selected from the group consisting of hydrogen, alkyl,C-carboxy, C-peptidyl and, combined, a five-member or 6-memberheteroalicyclic group containing at least one nitrogen; R¹⁰ is apolyhydroxyalkyl group; those of R⁴, R⁵, R⁶ and R⁷ which are notsubstituted as noted above are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido,N-Sulfonamido, trihalomethanesulfonyl, carbonyl, C-carboxy, O-carboxy,C-amido, N-amido, cyano, nitro, halo, O-thiocarbamyl, N-thiocarbamyl,guanyl and phosphonyl; and, R⁴ and R⁵ may combine to form a six-membercycloalkyl, heteroaryl or heteroalicyclic ring. and the physiologicallyacceptable salt and prodrugs thereof.
 2. The compound, salt or prodrugof claim 1 wherein R¹ is selected from the group consisting of hydrogenand alkyl.
 3. The compound, salt or prodrug of claim 2 wherein Z isoxygen.
 4. The compound, salt or prodrug of claim 3 wherein R² ishydrogen.
 5. The compound, salt or prodrug of claim 4 wherein A issulfur.
 6. The compound, salt or prodrug of claim 5 wherein R⁴ and R⁵,combined, form a six-member cycloalkyl ring.
 7. The compound, salt orprodrug of claim 6 wherein R⁶ is selected from the group consisting of—NR⁸R⁹, NR⁸R9— J(CH₂)_(m), —(CH₂)_(m)C(═Y) Q, —N═CNR⁸R⁹ and —NHR¹⁰. 8.The compound, salt or prodrug of claim 7 wherein R⁷ is selected from thegroup consisting of —NR⁸R⁹, —J(CH₂)_(m)—NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q,—N═CNR⁸R⁹ and —NHR¹⁰.
 9. The compound, salt or prodrug of claim 4wherein A is nitrogen.
 10. The compound, salt or prodrug of claim 9wherein R³ is hydrogen.
 11. The compound, salt or prodrug of claim 10wherein R⁴ and R⁵ are lower alkyl.
 12. The compound, salt or prodrug ofclaim 11 wherein R⁶ is selected from the groups consisting of —NR⁸R⁹,NP⁸R⁹—J(CH₂)_(m)—J(CH₂)_(m)C(═Y)Q. —N═CNR⁸R⁹ and —NHR¹⁰.
 13. Thecompound, salt or prodrug of claim 12 wherein R⁷ is selected from thegroup consisting of —NR⁸R⁹, NR⁸R⁹ —J(CH₂)_(m)—, —J(CH₂)_(m)C(═Y)Q,—N═CNR⁸R⁹ and —NHR¹⁰.
 14. The compound, salt or prodrug of claim 4wherein A and B are nitrogen.
 15. The compound, salt or prodrug of claim4 wherein A is oxygen.
 16. The compound, salt or prodrug of claim 4wherein A is nitrogen and B is sulfur.
 17. A pharmacological compositionof said compound, salt or prodrug of any one of claims 1-16.
 18. Amethod for treating or preventing a protein tyrosine kinase relateddisorder in an mammal comprising administering a therapeuticallyeffective amount of said pharmacological composition of claim 17 to saidmammal.
 19. The method of claim 18 wherein said protein tyrosine kinaserelated disorder comprises a cell proliferation, differentiation orgrowth disorder.
 20. The method of claim 19 wherein said cellproliferation, differentiation or growth disorder comprises a PDGF, EGF,IGF or MET related disorder.
 21. The method of claim 20 wherein saidPDGF related comprises blastoglioma, Kaposi's sarcoma, melanoma, lungcancer, ovarian cancer or prostate cancer.
 22. The method of claim 20wherein said EGF related disorder comprises squamous cell carcinoma,astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladdercancer.
 23. The method of claim 20 wherein said IGF related disordercomprises breast cancer, small-cell lung cancer, and gliomas.
 24. Themethod of claim 20 wherein said MET related disorder comprisescolorectal cancer, thyroid cancer, pancreatic and gastric carcinoma,leukemia and lymphoma, Hodgkin's disease and Burkitts disease.
 25. Themethod of claim 19 wherein protein tyrosine kinase related disordercomprises arthritis, diabetic retinopathy, restinosis, hepaticcirrhosis, atherosclerosis, glomerulonephritis, diabetic nephropathy,thrombic microangiopathy syndromes, transplant rejection, autoimmunedisease, diabetes or hyperimmune disorders.
 26. The method of claim 18wherein said mammal is a human.